Origin and distribution of enteric neurones in Xenopus.

Summary In Xenopus, we investigated the origin of enteric neurones and their distribution in relation to the extracellular matrix (ECM) components, fibronectin (FN) and tenascin (TN). Enteric neurone precursor cells originate from the anterior trunk neural crest (NC). They migrate along the ventromedial NC pathway (between somites and neural tube/notochord) into the primitive gut (via the dorsal mesentery/lateral plate mesoderm) where they differentiate into enteric neurones. NC cells were identified during their migration and in the gut using the X. laevis — X. borealis nuclear marker system. The neuronal character of NC cells in the gut could be demonstrated immunohistochemically with a monoclonal antibody against the HNK-1 epitope. This antibody is superior to N-CAM and neurofilament antibodies which proved insufficient in Xenopus.In early tadpoles (stage 45), enteric neurones occurred frequently in the mesenchymal lining of the oesophagus, either singly or in groups of two to three cells. In more distal portions of the digestive tract, enteric neurones were rarely found. In metamorphosing tadpoles (stage 62/63), enteric neurones were scattered singly beneath the mucosa, or formed small aggregates between the inner and outer muscle layer throughout the length of the digestive tract. The neurones occurred in positions corresponding to the myenteric and submucosal plexus of higher vertebrates.The distribution of enteric neurones was studied in relation to fibronectin (FN) and tenascin (TN), glycoproteins of the ECM, which support (FN) and inhibit (TN) amphibian NC cell migration. Using immunohisto-chemistry, FN was found during NC cell migration in ECM spaces along the ventromedial pathway, and in the gut between the mucosa and the muscle layers, where it would be able to support adhesion and migration of NC cells. TN, in contrast, appeared much later than FN, both in the dorsal trunk and also ventrally, in the gut. In older tadpoles, TN was present in the mesenchyme and muscle layers of the digestive tract, where it might have an inhibiting influence on the migration of enteric neurones within the gut wall.     

The thymus and tail regenerative capacity in Xenopus laevis tadpoles

A morphofunctional analysis of the thymus from differently aged Xenopus laevis tadpoles during regeneration of the tail is reported. In stage 50 larvae, competent to regenerate, the appendage cut provoked thymic structural modifications that affected the medullary microenvironment cells and changes in TNF-α immunoreactivity. Mucocyte-like cells, multicellular epithelial cysts, myoid cells and cells immunoreactive to TNF-α increased in number. Increased numbers of lymphocytes were also found in regenerating areas and, at the end of regeneration, thymic structural and immunocytochemical patterns were restored to control levels. The observed cellular responses and the induction of molecules critical for thymus constitutive processes suggest a stimulation of thymic function after tail amputation. In older larvae, whose capacity to form a new complete and correctly patterned tail was reduced, thymic morphological changes were more severe and may persist throughout the regeneration process with a significant reduction in organ size. In these larvae the histological patterns and the marked thymic decrease may be related to the events occurring during regeneration, i.e. the higher inflammatory response and the reduced tail regenerative potential.     

Ionic basis of resting membrane potential in frog taste cells

Intracellular recording of the resting membrane potential was made from taste cells of the bullfrog by replacing the interstitial fluid surrounding the cells by various physiological saline solutions. The resting potential of the taste cell was -28 +/- 4 mV (mean +/- S.E.) after replacement of the interstitial fluid by normal Ringer solution. The resting potential was very much dependent on the interstitial K+ concentration ([K+]). Tenfold increase in [K+]o decreased the resting potential by 18 mV. Total removal of interstitial Na+ increased the resting potential by 40%. Ouabain in a concentration of 0.5 nM decreased the resting potential by 36% possibly due to inhibition of the Na+-K+ exchange pump. Neither total removal nor fourfold increase of interstitial Ca2+ changed the resting potential. Complete elimination of interstitial Cl- did not change the resting potential. The mean permeability ratio PNa/PK was calculated to be 0.41. It is concluded that the resting potential of a frog taste cell consists of ionic and metabolic components, and that the ionic component is due to the permeability of the cell membrane to K+ and Na+.     

Ionic basis of the resting membrane potential and action potential in the pharyngeal muscle of Caenorhabditis elegans

The pharynx of C. elegans is a rhythmically active muscle that pumps bacteria into the gut of the nematode. This activity is maintained by action potentials, which qualitatively bear a resemblance to vertebrate cardiac action potentials. Here, the ionic basis of the resting membrane potential and pharyngeal action potential has been characterized using intracellular recording techniques. The resting membrane potential is largely determined by a K(+) permeability, and a ouabain-sensitive, electrogenic pump. As previously suggested, the action potential is at least partly dependent on voltage-gated Ca(2+) channels, as the amplitude was increased as extracellular Ca(2+) was increased, and decreased by L-type Ca(2+) channel blockers verapamil and nifedipine. Barium caused a marked prolongation of action potential duration, suggesting that a calcium-activated K(+) current may contribute to repolarization. Most notably, however, we found that action potentials were abolished in the absence of external Na(+). This may be due, at least in part, to a Na(+)-dependent pacemaker potential. In addition, the persistence of action potentials in nominally free Ca(2+), the inhibition by Na(+) channel blockers procaine and quinidine, and the increase in action potential frequency caused by veratridine, a toxin that alters activation of voltage-gated Na(+) channels, point to the involvement of a voltage-gated Na(+) current. Voltage-clamp analysis is required for detailed characterization of this current, and this is in progress. Nonetheless, these observations are quite surprising in view of the lack of any obvious candidate genes for voltage-gated Na(+) channels in the C. elegans genome. It would therefore be informative to re-evaluate the data from these homology searches, with the aim of identifying the gene(s) conferring this Na(+), quinidine, and veratridine sensitivity to the pharynx.     

The development of connections between the isthmic nucleus and the tectum in Xenopus and Limnodynastes tadpoles

In adult Anura reciprocal connections exist between the isthmic nucleus and ipsilateral tectum. These pathways, with a projection from the isthmic nucleus to the opposite tectum, constitute the binocular visual connections. Isthmic nuclei were examined histologically after injection of horseradish peroxidase into the tectum of Xenopus laevis and Limnodynastes dorsalis from midlarval stages to the completion of metamorphosis and in adults. The isthmic nuclei were present throughout, their cell number reaching the adult complement by metamorphic climax. At all stages each isthmic nucleus projected to both tecta. Late onset of electrical activity in the intertectal pathway cannot therefore be explained by the time at which isthmic fibres reach the tecta.     

The cellular basis of the convergence and extension of the Xenopus neural plate.

There is great interest in the patterning and morphogenesis of the vertebrate nervous system, but the morphogenetic movements involved in early neural development and their underlying cellular mechanisms are poorly understood. This paper describes the cellular basis of the early neural morphogenesis of Xenopus laevis. The results have important implications for neural induction. Mapping the fate map of the midneurula (Eagleson and Harris: J. Neurobiol. 21:427-440, 1990) back to the early gastrula with time-lapse video recording demonstrates that the prospective hindbrain and spinal cord are initially very wide and very short, and thus at the beginning of gastrulation all their precursor cells lie within a few cell diameters of the inducing mesoderm. In the midgastrula, the prospective hindbrain and spinal cord undergo very strong convergence and extension movements in two phases: In the first phase they primarily undergo thinning in the radial direction and lengthening (extension) in the animal-vegetal direction, and the second phase is characterized primarily by mediolateral narrowing (convergence) and anterior-posterior lengthening (extension). These movements also occur in sandwich explants of the gastrula, thus demonstrating the local autonomy of the forces producing them. Tracing cell movements with fluorescein dextran-labeled cells in embryos or explants shows that the initial thinning and extension occurs by radial intercalation of deep cells to form fewer layers of greater area, all of which is expressed as increased length. The subsequent convergence and extension occurs by mediolateral intercalation of deep cells to form a longer, narrower array. These results establish that a similar if not identical sequence of radial and mediolateral cell intercalations underlie convergence and extension of the neural and the mesoderm tissues (Wilson and Keller: Development, 112:289-300, 1991). Moreover, these results establish that radial and mediolateral intercalation are the principal neural cell behaviors induced by the planar signals emanating from the dorsal involuting marginal zone (the Spemann organizer) in the early gastrula (Keller et al: Develop. Dynamics, 193: 218-234, 1992). Radial and mediolateral intercalation are induced among the 5 to 7 rows of cells comprising the prospective hindbrain and spinal cord, thus producing the massive convergence and extension movements that narrow and elongate these regions of the nervous system in the late gastrula. A more general significance of these results is that neural induction is best analyzed and understood in terms of the dynamics of the morphogenetic processes involved.     

Regeneration of optic fibres through the chiasma in Xenopus laevis tadpoles

Summary The path through the chiasma followed by regenerating optic nerve fibres in Xenopus tadpoles was studied at light- and electron-microscopic levels, and with horseradish peroxidase as a fibre label. Over the period (5–10 days) in which regenerating fibres reach and cross the chiasma, they did not follow residual deep fibres through the chiasma, nor were they associated with the trail of degeneration in the chiasma which represented the remains of the deeper (older) parts of the original projection. The regenerating optic fibres were always seen in close association with the inner surfaces of the ependymoglial endfeet, or in the extracellular spaces that lie close to the endfeet in the most superficial part of the chiasma, where newly-growing fibres from the retinal margin are normally to be found.     

The slow inhibitory postsynaptic potential in rat hippocampal CA1 neurones is blocked by intracellular injection of QX-314

Intracellular recordings were made from CA1 pyramidal neurones in the rat hippocampus slice preparation. The recording electrodes contained potassium acetate (4 M) with or without the quaternary lidocaine derivative, QX-314 (50 mM). Both fast (f) and slow (s) inhibitory postsynaptic potentials (IPSP) were evoked by low-frequency orthodromic stimulation. The s-IPSP was rapidly reduced by QX-314 injection. It decreased along a similar time course to the dV/dt of the action potential (AP). The f-IPSP and excitatory postsynaptic potential were not significantly reduced in size at a time when the s-IPSP was virtually abolished by QX-314. It is concluded that conductance through the K⁺ channels which are coupled to GABA_B receptors is readily blocked by QX-314, while the Cl⁻ channels which are coupled to GABA_A receptors and the cation channels coupled to the glutamate receptors are relatively resistant to the local anaesthetic.     

The ionic mechanism of the slow outward current in Aplysia neurons

A slow outward current associated with spike frequency adaptation has been studied in the giant Aplysia neurons R2 and LP1. The current was observed during 60-s voltage clamp commands to potentials just below spike threshold. The slow outward current shows a marked voltage dependence at membrane potential less negative than -40 mV. The slow outward current is associated with increased membrane conductance. The K+ sensitivity of the slow outward current was studied by varying the extracellular K+ concentration and also by measuring potassium efflux with a K+-sensitive electrode. Both procedures indicated that the slow outward current was K+ dependent. Tail currents following the activation of the slow outward current were examined. They were shown to have a similar potassium sensitivity as the slow outward current and had a reversal potential near the potassium equilibrium potential for these cells. The sensitivity of the slow outward current to known blockers of K+ currents, tetraethylammonium and 4-aminopyridine, was tested. The sensitivity was much less than that reported for other K+ currents. The sensitivity of the slow outward current to changes of the extracellular concentrations of Na+ and Cl- ions, as well as electrogenic pump inhibitors, was tested. The results indicate that the slow outward current is much less sensitive to these changes than to the manipulations of the extracellular K+ ion concentration. We tested the sensitivity of this current to manipulations of intracellular and extracellular Ca2+ ion concentrations. We found that the current persisted at a slightly reduced level in the absence of extracellular calcium or in the presence of calcium blocking agents, cobalt and lanthanum. Intracellular injection of the calcium chelator EGTA at a concentration sufficient to block the Ca2+-dependent K+ current, seen after a brief (1.4-s) burst of action potentials, had minimal effects on the slow outward current. Procedures thought to increase intracellular Ca2+ were tested. We found that exposure of the cell to solutions containing elevated Ca2+ concentrations for prolonged periods increased the slow outward current. Also, treatment with drugs thought to elevate intracellular Ca2+ increased the slow outward current. In conclusion, the slow outward current results from an increased K+ conductance.(ABSTRACT TRUNCATED AT 400 WORDS)     

A riluzole- and valproate-sensitive persistent sodium current contributes to the resting membrane potential and increases the excitability of sympathetic neurones

Non-adapting superior cervical ganglion (SCG) neurones with a clustering activity and sub-threshold membrane potential oscillations were occasionally recorded, suggesting the presence of a persistent sodium current (I _NaP). The perforated-patch technique was used to establish its properties and physiological role. Voltage-clamp experiments demonstrated that all SCG cells have a TTX-sensitive I _NaP activating at about −60 mV and with half-maximal activation at about −40 mV. The mean maximum I _NaP amplitude was around −40 pA at −20 mV. Similar results were achieved when voltage steps or voltage ramps were used to construct the current–voltage relationships, and the general I _NaP properties were comparable in mouse and rat SCG neurons. I _NaP was inhibited by riluzole and valproate with an IC₅₀ of 2.7 and 3.8 μM, respectively, while both drugs inhibited the transient sodium current (I _NaT) with a corresponding IC₅₀ of 34 and 150 μM. It is worth noting that 30 μM valproate inhibited the I _NaP by 70% without affecting the I _NaT. In current clamp, valproate (30 μM) hyperpolarised resting SCG membranes by about 2 mV and increased the injected current necessary to evoke an action potential by about 20 pA. Together, these results demonstrate for the first time that a persistent sodium current exists in the membrane of SCG sympathetic neurones which could allow them to oscillate in the sub-threshold range. This current also contributes to the resting membrane potential and increases cellular excitability, so that it is likely to play an important role in neuronal behaviour. J. A. L. and M. R. have contributed equally to this work.     

Isolation and characterization of slow, depolarizing responses of cardiac ganglion neurons in the crab, Portunus sanguinolentus.

1. Tetrodotoxin-resistant, active responses to depolarization of the large cardiac ganglion cells were studied in semi-isolated preparations from the crab, Portunus sanguinolentus. Impulse activity was monitored with extracellular electrodes, simultaneous recordings from two or three large cells were made with intracellular electrodes, and current was passed via a bridge or second intracellular electrode. Preparations were continuously perfused with saline containing 3 x 10(-7) M tetrodotoxin (TTX). 2. About 20 min after introduction of TTX, small-cell impulses and resultant EPSPs in large cells cease, while rhythmic, spontaneous bursting of large cells continues. A pacemaker depolarization between bursts and slow depolarizations underlying the impulse bursts are prominent at this time. Shortly after, spontaneous burst rate slows, and at ca. 25 min, the ganglion becomes electrically quiescent. 3. In the quiescent, TTX-perfused ganglion, injection of depolarizing current into any one of the large cells results in active responses. At current strengths of sufficient intensity and duration (e.g., 20 nA, 20 ms; 5 nA, 500 ms) to depolarize a large cell by ca. 10 mV from resting potential (-53 mV, avg), the graded responses become regenerative and of constant form, provided the stimulation rate is less thna 0.15/s. Such responses have been termed "driver potentials." At more rapid rates, thresholds are increased and responses reduced. 4. Driver potentials of anterior large cells reach peak amplitudes of ca. 20 mV (to -32 mV), have maximum rates of rise of 0.45 V/s and of fall of 0.2 V/s, and a duration of ca. 250 ms. They are followed by hyperpolarizing afterpotentials, a rapidly decaying one (1 s) to -58 mV, followed by a slowly decaying one (7.5 s), -55 mV. Responses of posterior large cells are smaller (16 mV) and slower; the site of active response may be at a distance from the soma. 5. The ability of elicit near-synchronous responses and the identity of amplitude and form of responses among anterior cells and of posterior cells, regardless of which cell receives depolarizing current, indicates that all cells undergo active responses and are stimulated by electrotonic spread of depolarization. 6. The responses involve a conductance increase since memses during a driver potential are much reduced. 7. Depolarization by steady current increases the absolute threshold, decreases the maximum depolarization of the peak, and slows rates of rise and fall. Hyperpolarization increases rates of rise and fall; the absolute value reached by the peak depolarization is unchanged. Hyperpolarization reduces the amplitude of the rapid after-potential relative to the displaced resting potential. 8. Hyperpolarizing current pulses imposed during the rise and peak of driver-potential responses are followed by redevelopment of a complete response. Sufficiently strong hyperpolarization can terminate a response. The current strength needed to terminate a response decreases the later during the response the pulse is given...     

Complex slow potential generators in a simplified attention paradigm.

We have recently obtained evidence for complex multifocal, individually variable generators of slow cortical potentials, elicited during performance of visual tasks involving expecting attention, comparison and memory [Basile, L.F.H., Ballester, G., Castro, C.C., and Gattaz, W.F., 2002. Multifocal slow potential generators revealed by high-resolution EEG and current density reconstruction. Int. J. Psychophysiol., 45 (3), 227-240; Basile, L.F.H, Baldo, M.V., Castro, C.C., and Gattaz, W.F. 2003. The generators of slow potentials obtained during verbal, pictorial and spatial tasks. Int. J. Psychophysiol., 48, 55-65]. The cue-target aspect of traditional paradigms for attention studies is equivalent to 'warning S1'-'imperative S2' in slow potential designs. We simplified Posner's spatial cueing task [Posner, M.I. 1980. Orienting of attention.Q. J. Exp. Psychol. Feb;32 (1), 3-25; Posner, M.I., Snyder, C.R., Davidson, B.J. 1980. Attention and the detection of signals. J Exp Psychol. Jun; 109 (2), 160-174] to temporal cuing only, by using visual cues to indicate the mere presence, on a known central position, of the eventual target (17 ms duration, +/-0.3 degrees grey circle). We recorded slow potentials on 12 healthy subjects, by 124-channel EEG system (Neuroscan Inc.), and modeled their generators using current density reconstruction (CDR) by L(p) 1.2 norm minimization ("Curry V4.6", Neurosoft Inc.) applied to the target onset time. MRIs were obtained for each subject for constraining source models to individual brain anatomy. Average slow potentials were computed from above 60 artifact-free EEG-epochs (ISI=1.6 s, average ITI=2.5 s). We tabulated individual cortical current distributions by cytoarchitectonic area of Brodmann, after scaling into negligible, low, moderate and strong local density, based on percentile bands with respect to absolute maximum current. Despite the task's simplicity, the main result was individual variability and complexity in both scalp voltage and cortical current distributions. As observed in our previous studies, there was strong intersubject variability in the exact distribution of task-related cortical activity. Only parietal area 7 bilaterally was non-negligibly active in all subjects (currents above 10% maximum). As opposed to drawing conclusions based on group averaged data, we propose that activity by cytoarchitectonic area be ranked and statistically analysed only after being scaled on each individual. Based on the present results, the concept of a universal attention-related set of cortical areas if restricted to common areas across subjects is challenged, since even area 7 may no longer be common when the sample size becomes larger. We discuss the fact that group averaging may de-emphasize weakly but consistently active areas, and emphasize strongly but inconsistently active ones.