Análisis: factor de impacto y comunicación científica / Analysis: impact factor and scientific communication

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Réplica del Comité Ejecutivo. SIP30-AI: reflexiones / SIP30-AI: reflections. Reply of the Executive Committee

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Año nuevo, vida nueva. Nuevos directores de áreas temáticas en Revista de Neurología / New year, new life. New subject editors at Revista de Neurología

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Neurología de la Conducta / Behavioral neurology

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Effects of fluctuations on electrical properties of gap-junction connected cells in the turtle retina.

Electrical properties of gap-junction connected cells (input voltage and length constant) are shown to depend strongly on fluctuations in membrane and contact conductances. This opens new possibilities and incorporates a further difficulty to the analysis of electrophysiological data, since four, instead of two, parameters (the average values and the magnitude of fluctuations of the two conductances) have to be used in fitting the experimental data. The discussion is illustrated by investigating the effects of dopamine on signal spreading in horizontal cells of turtle retina, assuming a linear cell arrangement. It is shown that while a standard fitting with the average values of the two conductances leads to the conclusion that both are equally affected by dopamine, including fluctuations allows fitting the data by varying just the average contact conductance plus the magnitude of fluctuations.

On the validity of the two-cells model in the analysis of passive electrical properties of gap-junction connected cells.

It is a rather extended practice to derive electrophysiological data (membrane and contact conductances) from experimental data for gap-junction tissues assuming that electrical connections are reduced to cell pairs. It is here shown that, if the length constant is sufficiently large, the mentioned procedure can lead to qualitatively incorrect results.

La Neurociencia en el cambio de siglo / Neuroscience at the turn of the century

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Junctional communication of pancreatic beta cells contributes to the control of insulin secretion and glucose tolerance.

Proper insulin secretion requires the coordinated functioning of the numerous beta cells that form pancreatic islets. This coordination depends on a network of communication mechanisms whereby beta cells interact with extracellular signals and adjacent cells via connexin channels. To assess whether connexin-dependent communication plays a role in vivo, we have developed transgenic mice in which connexin 32 (Cx32), one of the vertebrate connexins found in the pancreas, is expressed in beta cells. We show that the altered beta-cell coupling that results from this expression causes reduced insulin secretion in response to physiologically relevant concentrations of glucose and abnormal tolerance to the sugar. These alterations were observed in spite of normal numbers of islets, increased insulin content, and preserved secretory response to glucose by individual beta cells. Moreover, glucose-stimulated islets showed improved electrical synchronization of these cells and increased cytosolic levels of Ca(2+). The results show that connexins contribute to the control of beta cells in vivo and that their excess is detrimental for insulin secretion.

Engineering pancreatic islets.

Pancreatic islets are neuroendocrine organs that control blood glucose homeostasis. The precise interplay of a heterogeneous group of cell populations (beta, alpha, delta and PP cells) results in the fine-tuned release of counterbalanced hormones (insulin, glucagon, somatostatin and pancreatic polypeptide respectively). Under the premises of detailed knowledge of the physiological basis underlying this behaviour, two lines of investigation might be inferred: generating computational and operational models to explain and predict this behaviour and engineering islet cells to reconstruct pancreatic endocrine function. Whilst the former is being fuelled by new computational strategies, giving biophysicists the possibility of modelling a system in which new "emergent" properties appear, the latter is benefiting from the useful tools and strategic knowledge achieved by molecular, cell and developmental biologists. This includes using tumour cell lines, engineering islet cell precursors, knowledge of the mechanisms of differentiation, regeneration and growth and, finally, therapeutic cloning of human tissues. Gaining deep physiological understanding of the basis governing these processes is instrumental for engineering new pancreatic islets.

Heterogeneous populations of cells mediate spontaneous synchronous bursting in the developing hippocampus through a frequency-dependent mechanism.

Under normal conditions, hippocampal slices from newborn rats and rabbits (postnatal days 0-8) show spontaneous synchronous bursts known as giant depolarizing potentials. These bursts are recorded from CA3, CA1 and the fascia dentata in both intact slices and isolated hipocampal regions. Giant depolarizing potentials are network-driven events resulting from the synergistic activation of N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxadepropionate and GABA(A) receptors, the latter playing an excitatory role. Recently, we showed that they spontaneously emerge in an all-or-none manner after the increase of synaptic and cellular activity beyond a threshold frequency [Menendez de la Prida L. and Sanchez-Andres J. V. (1999) J. Neurophysiol. 82, 202-208]. Under this framework, background levels of spontaneous activity at individual neurons build up network synchronization 100-300ms prior to the onset of giant depolarizing potentials. However, the role of distinct cellular populations and connectivity in determining the threshold frequency has not been examined. By performing simultaneous intracellular recordings from pyramidal cells, non-pyramidal cells and interneurons, we investigated their participation in the generation of giant depolarizing potentials. Electrodes containing Neurobiotin were used to examine the cellular morphology. We found that giant depolarizing potentials were not initiated from a single pacemaker cellular group; instead, they involved recurrent cooperation among these groups, which contributed differently according to their intrinsic firing capability. In all the neurons examined, the onset of these bursts took place in an all-or-none frequency-dependent manner, both spontaneously (depending on the frequency of the excitatory postsynaptic potentials) or when triggered by extracellular stimulation. The CA3 threshold of frequency was at 12Hz in both pyramidal cells and interneurons, while in the fascia dentata it was 17Hz. The application of 6-cyano-7-nitroquinoxaline-2,3-dione increased CA3 threshold of frequency up to 50Hz, suggesting that it is determined by combined synaptic components. We examined the role of postsynaptic summation on the threshold of frequency. Heterogeneity is present among the cellular groups, pyramidal neurons from CA1 and CA3 showing less evidence of postsynaptic summation prior to giant depolarizing potentials. Cells showing stronger evidence of postsynaptic summation were more typically recorded at the hilus, the granule layer of the fascia dentata and the CA3/CA4 area. Nevertheless, for a given cell, not all the giant depolarizing potentials were preceded by summation of postsynaptic potentials. These outcomes, together with the long and variable time delays recorded between different areas, strongly suggest that giant depolarizing potentials are locally generated from different initiation sites and not from a single region. We discuss these results in view of the principles underlying hyperexcitability in hippocampal slices, i.e. the intrinsic firing properties of individual cells and the connectivity patterns.

Role of architecture in determining passive electrical properties in gap junction-connected cells.

The electrical properties of gap junction-connected cells were analysed in terms of their architectural organization. Two major architectural categories were considered: trees and rings. Trees are described by means of Bethe lattices (lattices with no rings) with arbitrary co-ordination and rings by two-dimensional periodic lattices with fourfold (square) or sixfold (triangular) co-ordination. The Bethe lattice is solved analytically by the transfer constant method, which allows the introduction of several physiologically relevant effects in a very simple manner. The experimental data for the length constant and the input resistance were fitted by varying the coupling and membrane resistances for various morphologies. The large variations in the length constant observed experimentally in two systems (turtle retina horizontal cells with and without dopamine and pancreatic beta-cells in the active and silent phases) could not be explained by means of the Bethe lattice, indicating that the cell arrangements form rings. Subsequent analysis by means of a linear chain and the square and triangular lattices showed the crucial relevance of architecture in deriving the electrical characteristics of gap junction-connected cells from experimental data.

Effects of calcium buffering on glucose-induced insulin release in mouse pancreatic islets: an approximation to the calcium sensor.

1. The properties of the calcium sensor for glucose-induced insulin secretion have been studied using cell-permeant Ca2+ buffers with distinct kinetics and affinities. In addition, submembrane cytosolic Ca2+ distribution has been modelled after trains of glucose-induced action potential-like depolarizations. 2. Slow Ca2+ buffers (around 1 mmol l-1 intracellular concentration) with different affinities (EGTA and Calcium Orange-5N) did not significantly affect glucose-induced insulin release. Modelling showed no effect on cytosolic Ca2+ concentrations at the outermost shell (0.05 microm), their effects being observed in the innermost shells dependent on Ca2+ affinity. 3. In contrast, fast Ca2+ buffers (around 1 mmol l-1 intracellular concentration) with different affinities (BAPTA and Calcium Green-5N) caused a 50 % inhibition of early insulin response and completely blocked the late phase of glucose-induced insulin response, their simulations showing a decrease of [Ca2+]i at both the inner and outermost shells. 4. These data are consistent with the existence in pancreatic beta-cells of a higher affinity Ca2+ sensor than that proposed for neurons. Moreover, these data are consistent with the proposed existence of two distinct pools of granules: (i) 'primed' vesicles, colocalized with Ca2+ channels and responsible of the first phase of insulin release; and (ii) 'reserved pool' vesicles, not colocalized and responsible for the second phase.

Mechanisms of glucose hypersensitivity in beta-cells from normoglycemic, partially pancreatectomized mice.

Increased beta-cell sensitivity to glucose precedes the loss of glucose-induced insulin secretion in diabetic animals. Changes at the level of beta-cell glucose sensor have been described in these situations, but it is not clear whether they fully account for the increased insulin secretion. Using a euglycemic-normolipidemic 60% pancreatectomized (60%-Px) mouse model, we have studied the ionic mechanisms responsible for increased beta-cell glucose sensitivity. Two weeks after Px (Px14 group), Px mice maintained normoglycemia with a reduced beta-cell mass (0.88 +/- 0.18 mg) compared with control mice (1.41 +/- 0.21 mg). At this stage, the dose-response curve for glucose-induced insulin release showed a significant displacement to the left (P < 0.001). Islets from the Px14 group showed oscillatory electrical activity and cytosolic Ca2+ ([Ca2+]i) oscillations in response to glucose concentrations of 5.6 mmol/l compared with islets from the control group at 11.1 mmol/l. All the above changes were fully reversible both in vitro (after 48-h culture of islets from the Px14 group) and in vivo (after regeneration of beta-cell mass in islets studied 60 days after Px). No significant differences in the input resistance and ATP inhibition of ATP-sensitive K+ (K(ATP)) channels were found between beta-cells from the Px14 and control groups. The dose-response curve for glucose-induced MTT (C,N-diphenyl-N''-4,5-dimethyl thiazol 2 yl tetrazolium bromide) reduction showed a significant displacement to the left in islets from the Px14 group (P < 0.001). These results indicate that increased glucose sensitivity in terms of insulin secretion and Ca2+ signaling was not due to intrinsic modifications of K(ATP) channel properties, and suggest that the changes are most likely to be found in the glucose metabolism.

Nonlinear frequency-dependent synchronization in the developing hippocampus.

Synchronous population activity is present both in normal and pathological conditions such as epilepsy. In the immature hippocampus, synchronous bursting is an electrophysiological conspicuous event. These bursts, known as giant depolarizing potentials (GDPs), are generated by the synchronized activation of interneurons and pyramidal cells via GABAA, N-methyl-D-aspartate, and AMPA receptors. Nevertheless the mechanism leading to this synchronization is still controversial. We have investigated the conditions under which synchronization arises in developing hippocampal networks. By means of simultaneous intracellular recordings, we show that GDPs result from local cooperation of active cells within an integration period prior to their onset. During this time interval, an increase in the number of excitatory postsynaptic potentials (EPSPs) takes place building up full synchronization between cells. These EPSPs are correlated with individual action potentials simultaneously occurring in neighboring cells. We have used EPSP frequency as an indicator of the neuronal activity underlying GDP generation. By comparing EPSP frequency with the occurrence of synchronized GDPs between CA3 and the fascia dentata (FD), we found that GDPs are fired in an all-or-none manner, which is characterized by a specific threshold of EPSP frequency from which synchronous GDPs emerge. In FD, the EPSP frequency-threshold for GDP onset is 17 Hz. GDPs are triggered similarly in CA3 by appropriate periodic stimulation of mossy fibers. The frequency threshold for CA3 GDP onset is 12 Hz. These findings clarify the local mechanism of synchronization underlying bursting in the developing hippocampus, indicating that GDPs are fired when background levels of EPSPs or action potentials have built up full synchronization by firing at specific frequencies (>12 Hz). Our results also demonstrate that spontaneous EPSPs and action potentials are important for the initiation of synchronous bursts in the developing hippocampus.

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices.

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices. Giant depolarizing potentials (GDPs) are generated by the interplay of the depolarizing action of GABA and glutamate. In this study, single and dual whole cell recordings (in current-clamp configuration) were performed from CA3 pyramidal cells in hippocampal slices obtained from postnatal (P) days P1- to P6-old rats to evaluate the role of ionotropic glutamate receptors in GDP generation. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-40 microM) completely blocked GDPs. However, in the presence of CNQX, it was still possible to re-induce the appearance of GDPs with GABA (20 microM) or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) (5 microM). This effect was prevented by the more potent and selective AMPA receptor antagonist GYKI 53655 (50-100 microM). In the presence of GYKI 53655, both kainic or domoic acid (0.1-1 microM) were unable to induce GDPs. In contrast, bath application of D-(-)-2-amino-5-phosphonopentanoic acid (50 microM) or (+)-3-(2carboxy-piperazin-4-yl)-propyl-L-phosphonic acid (20 microM) produced only a 37 +/- 9% (SE) and 36 +/- 11% reduction in GDPs frequency, respectively. Cyclothiazide, a selective blocker of AMPA receptor desensitization, increased GDP frequency by 76 +/- 14%. Experiments were also performed with an intracellular solution containing KF to block GABAA receptor-mediated responses. In these conditions, a glutamatergic component of GDP was revealed. GDPs could still be recorded synchronous with those detected simultaneously with KCl-filled electrodes, although their amplitude was smaller. Similar results were found in pair recordings obtained from minislices containing only a small portion of the CA3 area. These data suggest that GDP generation requires activation of AMPA receptors by local release of glutamate from recurrent collaterals.