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R Yuste

Publications and source records attributed to R Yuste.

At least 19 recordsLinked to original sources
PubMed2001

Dynamics of spontaneous activity in neocortical slices.

The flow of activity in the cortical microcircuitry is poorly understood. We use calcium imaging to reconstruct, with millisecond and single-cell resolution, the spontaneous activity of populations of neurons in unstimulated slices from mouse visual cortex. We find spontaneous activity correlated among networks of layer 5 pyramidal cells. Synchronous ensembles occupy overlapping territories, often share neurons, and are repeatedly activated. Sets of neurons are also sequentially activated numerous times. Network synchronization and sequential correlations are blocked by glutamatergic antagonists, even though spontaneous firing persists in many "autonomously active" neurons. This autonomous activity is periodic and depends on hyperpolarization-activated cationic (H) and persistent sodium (Na(p)) currents. We conclude that the isolated neocortical microcircuit generates spontaneous activity, mediated by a combination of intrinsic and circuit mechanisms, and that this activity can be temporally precise.

Action Potentials↗
PubMed2001

Topology of gap junction networks in C. elegans.

Gap junctions are prevalent in every nervous system, but their role in information processing remains largely unknown. In C. elegans, the role of gap junctional communication in touch sensitivity has been demonstrated. In this animal, the entire complement of gap junctions in the nervous system is documented, therefore providing a good model for the computational investigation of circuit functions of gap junctions.We explored several hypotheses about the role of gap junctions in the nervous system of C. elegans by systematically analysing an anatomical database with recursive algorithms. We find that gap junctions connect different sets of neurons from those connected by chemical synapses. In addition, when analysing the topology of the gap-junction networks, we find that, surprisingly, most (92%) neurons in the worm are linked in a single gap-junction network. The worm nervous system can only be divided into smaller networks by assuming that two or more gap junctions are necessary for functional coupling or that neural activity has limited propagation. However, these groups, and others identified using algorithms with subsets or combinations of restrictive criteria, do not correspond to any known circuits identified in genetic and behavioral studies. Finally, we notice that the function of some gap junctions appears linked to their precise location on the neuronal processes. We propose that the location of the gap junctions within the neuron determines their functional role.

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PubMed2001

Calcium imaging of epileptiform events with single-cell resolution.

Epileptic discharges propagate through apparently normal circuits, although it is still unclear how this recruitment takes place. To understand the role of different classes of neurons in neocortical epilepsy, we have developed a novel imaging assay that detects which neurons participate in epileptiform discharges. Using calcium imaging of neuronal populations during bicuculline-induced spontaneous epileptiform events in slices from juvenile mouse somatosensory cortex, we find that fast calcium transients correlate with epileptiform field potentials and intracellular depolarizing shifts and can be used as an optical signature that a given neuron has participated in an epileptiform event. Our results demonstrate a novel method to characterize epileptiform events with single-cell resolution. In addition, our data are consistent with an important role for layer 5 in generating neocortical seizures and indicate that subgroups of neurons are particularly prone to epileptiform recruitment.

Action Potentials↗
PubMed2001

Stereotyped position of local synaptic targets in neocortex.

The microcircuitry of the mammalian neocortex remains largely unknown. Although the neocortex could be composed of scores of precise circuits, an alternative possibility is that local connectivity is probabilistic or even random. To examine the precision and degree of determinism in the neocortical microcircuitry, we used optical probing to reconstruct microcircuits in layer 5 from mouse primary visual cortex. We stimulated "trigger" cells, isolated from a homogenous population of corticotectal pyramidal neurons, while optically detecting "follower" neurons directly driven by the triggers. Followers belonged to a few selective anatomical classes with stereotyped physiological and synaptic responses. Moreover, even the position of the followers appeared determined across animals. Our data reveal precisely organized cortical microcircuits.

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PubMed2001

Morphological changes in dendritic spines associated with long-term synaptic plasticity.

Dendritic spines are morphological specializations that receive synaptic inputs and compartmentalize calcium. In spite of a long history of research, the specific function of spines is still not well understood. Here we review the current status of the relation between morphological changes in spines and synaptic plasticity. Since Cajal and Tanzi proposed that changes in the structure of the brain might occur as a consequence of experience, the search for the morphological correlates of learning has constituted one of the central questions in neuroscience. Although there are scores of studies that encompass this wide field in many species, in this review we focus on experimental work that has analyzed the morphological consequences of hippocampal long-term potentiation (LTP) in rodents. Over the past two decades many studies have demonstrated changes in the morphology of spines after LTP, such as enlargements of the spine head and shortenings of the spine neck. Biophysically, these changes translate into an increase in the synaptic current injected at the spine, as well as shortening of the time constant for calcium compartmentalization. In addition, recent online studies using time-lapse imaging have reported increased spinogenesis. The currently available data show a strong correlation between synaptic plasticity and morphological changes in spines, although at the same time, there is no evidence that these morphological changes are necessary or sufficient for the induction or maintenance of LTP. Still, they highlight once more how form and function go hand in hand in the central nervous system.

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PubMed2001

On the function of dendritic spines.

Dendritic spines occupy a strategic position in the central nervous system, yet their function is still under debate. Over the past decades, many hypotheses have been put forward to explain the specific function of spines. Recently, imaging experiments have demonstrated that spines compartmentalize calcium, a role that appears necessary for input-specific forms of synaptic plasticity. In addition, it has been discovered that spine morphology is plastic over fast time scales and can be controlled by specific biochemical pathways. Also, several aspects of the spine's morphology appear to be intricately linked to its function. The authors review these recent data and incorporate them into a model for the function of dendritic spines in CNS circuits. In their proposal, spines serve to specifically connect sparse inputs and therefore minimize the wiring necessary in the CNS while maximizing connectivity. By virtue of the same design, spines isolate inputs and thus implement local learning rules. These rules appear only necessary with sparse inputs so these two functions are intimately related. Spines therefore would play a crucial circuit role, remarkably analogous to synaptic matrix elements of associative neural networks. This model highlights the economical, yet elegant, design of CNS circuits.

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PubMed2000

Regulation of spine calcium dynamics by rapid spine motility.

Dendritic spines receive most excitatory inputs in the CNS and compartmentalize calcium. Spines also undergo rapid morphological changes, although the function of this motility is still unclear. We have investigated the effect of spine movement on spine calcium dynamics with two-photon photobleaching of enhanced green fluorescent protein and calcium imaging of action potential-elicited transients in spines from layer 2/3 pyramidal neurons in mouse visual cortex slices. The elongation or retraction of the spine neck during spine motility alters the diffusional coupling between spine and dendrite and significantly changes calcium decay kinetics in spines. Our results demonstrate that the spine's ability to compartmentalize calcium is constantly changing.

Acoustic Stimulation↗
PubMed2000

Optical probing of neuronal circuits with calcium indicators.

An experimental difficulty in unraveling circuits in the mammalian nervous system is the identification of postsynaptic targets of a given neuron. Besides ultrastructural reconstructions, simultaneous recordings from pairs of cells in brain slices have been used to identify connected neurons. We describe in this paper a technique using calcium imaging that allows rapid identification of potential postsynaptic targets. This method consists of stimulating one neuron ("trigger") while imaging a population of cells to detect which other neurons ("followers") are activated by the trigger. By using bulk-loading of calcium indicators in slices of mouse visual cortex, we demonstrate that neurons that display somatic calcium transients time-locked to the spikes of a trigger neuron can be monosynaptically connected to it. This technique could be applied to reconstruct and assay circuits in the central nervous system.

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PubMed2000

Mechanisms of calcium decay kinetics in hippocampal spines: role of spine calcium pumps and calcium diffusion through the spine neck in biochemical compartmentalization.

Dendritic spines receive most excitatory inputs in the CNS and compartmentalize calcium. Although the mechanisms of calcium influx into spines have been explored, it is unknown what determines the calcium decay kinetics in spines. With two-photon microscopy we investigate action potential-induced calcium dynamics in spines from rat CA1 pyramidal neurons in slices. The [Ca(2+)](i) in most spines shows two decay kinetics: an initial fast component, during which [Ca(2+)](i) in spines decays to dendritic levels, followed by a slower decay phase in which the spine follows dendritic kinetics. The correlation between [Ca(2+)](i) in spine and dendrite at the breakpoint of the decay kinetics demonstrates diffusional equilibration between spine and dendrite during the slower component. To explain the faster initial decay, we rule out saturation or kinetic effects of endogenous or exogenous buffers and focus instead on (1) active calcium extrusion and (2) buffered diffusion of calcium from spine to dendrite. The presence of an undershoot in most spines indicates that extrusion mechanisms can be intrinsic to the spine. Supporting the two mechanisms, pharmacological blockade of smooth endoplasmic reticulum calcium (SERCA) pumps and the length of the spine neck affect spine decay kinetics. Using a mathematical model, we find that the contribution of calcium pumps and diffusion varies from spine to spine. We conclude that dendritic spines have calcium pumps and that their density and kinetics, together with the morphology of the spine neck, determine the time during which the spine compartmentalizes calcium.

Action Potentials↗
PubMed2000

Analysis of spine morphological plasticity in developing hippocampal pyramidal neurons.

Dendritic spines are targets of most excitatory inputs in the central nervous system (CNS) and are morphologically heterogeneous. Ultrastructural studies have traditionally classified spines into four major categories (filopodia, stubby, thin, and mushroom) based on their distinct morphologies. The recent discovery of rapid morphological plasticity of spines has raised the possibility that those categories, rather than being intrinsically different populations of spines, represent instead temporal snapshots of a single dynamic phenomenon. We examined this question with two-photon time-lapse imaging of developing hippocampal pyramidal neurons, transfected with E-GFP in cultured slices. After blind scoring to morphologically classify spines into the four traditional groups, we analyzed the fate of populations of spines over a period of 2-4 h. We found considerable morphological conversions among all categories, although systematic trends were detected. While most stubbies and spines (defined for our analysis as the combination of thin and mushroom protrusions) retained their basic morphologies, most filopodia transformed into stubbies and spines, although they could also extend out of existing spines. Our results suggest that in developing hippocampal pyramidal neurons, traditional morphological distinctions are stable over short (<4 h) periods of time, but that at the same time, considerable mixing among these groups takes place.

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PubMed2000

A custom-made two-photon microscope and deconvolution system.

We describe in detail a custom-built two-photon microscope based on a modified confocal scanhead (Olympus Fluoview) and mode-locked Ti:sapphire laser (Coherent Mira 900). This system has internal detectors as well as external whole-field detection and an electrooptical modulator for blanking the beam on flyback and effecting fast changes in excitation intensity. This microscope can be used in deep, scattering samples for quantitative measurements with a wide range of fluorophores (GFP, fura, calcium green, calcium orange, fluo-3, DiI, DiO, fluorescein, rhodamine), for fluorescent photobleaching recovery and for uncaging. Images obtained with this system can be deconvolved with the Estimation Maximization algorithm using the program XCOSM (freeware available at: http://www.ibc.wustl.edu/bcl/ xcosm/).

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PubMed2000

From form to function: calcium compartmentalization in dendritic spines.

Dendritic spines compartmentalize calcium, and this could be their main function. We review experimental work on spine calcium dynamics. Calcium influx into spines is mediated by calcium channels and by NMDA and AMPA receptors and is followed by fast diffusional equilibration within the spine head. Calcium decay kinetics are controlled by slower diffusion through the spine neck and by spine calcium pumps. Calcium release occurs in spines, although its role is controversial. Finally, the endogenous calcium buffers in spines remain unknown. Thus, spines are calcium compartments because of their morphologies and local influx and extrusion mechanisms. These studies highlight the richness and heterogeneity of pathways that regulate calcium accumulations in spines and the close relationship between the morphology and function of the spine.

Animals↗
PubMed2000

Regulation of dendritic spine morphology by the rho family of small GTPases: antagonistic roles of Rac and Rho.

Dendritic spines mediate most excitatory transmission in the mammalian CNS and have been traditionally considered stable structures. Following the suggestion that spines may 'twitch', it has been recently shown that spines are capable of rapid morphological rearrangements. Because of the role of the small GTPases from the Rho family in controlling neuronal morphogenesis, we investigated the effects of several members of this biochemical signaling pathway in the maintenance of the morphology of extant dendritic spines by combining biolistic transfection of pyramidal neurons in cultured cortical and hippocampal slices with two-photon microscopy. We find a variety of effects on the density and morphology of dendritic spines by expressing either constitutively active or dominant negative forms of several small GTPases of the Rho family, by blocking the entire pathway with Clostridium difficile toxin B or by blocking Rho with C3 transferase. We propose a model where Rac promotes spine formation, while Rho prevents it. We conclude that the small GTPases provide antagonistic control mechanisms of spine maintenance in pyramidal neurons.

Animals↗
PubMed2000

Aberrant development of hippocampal circuits and altered neural activity in netrin 1-deficient mice.

Diffusible factors, including netrins and semaphorins, are believed to be important cues for the formation of neural circuits in the forebrain. Here we have examined the role of netrin 1 in the development of hippocampal connections. We show that netrin 1 and its receptor, Dcc, are expressed in the developing fimbria and in projection neurons, respectively, and that netrin 1 promotes the outgrowth of hippocampal axons in vitro via DCC receptors. We also show that the hippocampus of netrin 1-deficient mice shows a misorientation of fiber tracts and pathfinding errors, as detected with antibodies against the surface proteins TAG-1, L1 and DCC. DiI injections show that hippocampal commissural axons do not cross the midline in these mutants. Instead, when axons approach the midline, they turn ventrally and form a massive aberrant projection to the ipsilateral septum. In addition, both the ipsilateral entorhino-hippocampal and the CA3-to-CA1 associational projections show an altered pattern of layer-specific termination in netrin 1-deficient mice. Finally, optical recordings with the Ca(2+) indicator Fura 2-AM show that spontaneous neuronal activity is reduced in the septum of netrin 1-mutant mice. We conclude that netrin 1 is required not only for the formation of crossed connections in the forebrain, but also for the appropriate layer-specific targeting of ipsilateral projections and for the control of normal levels of spontaneous neural activity.

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PubMed1999

Involvement of cajal-retzius neurons in spontaneous correlated activity of embryonic and postnatal layer 1 from wild-type and reeler mice.

Cajal-Retzius (CR) cells are a transient population of neurons in developing cortical layer 1 that secrete reelin, a protein necessary for cortical lamination. Combining calcium imaging of cortical hemispheres and cross-correlation analysis, we previously found spontaneous correlated activity among non-CR neurons in postnatal rat layer 1. This correlated activity was blocked by GABAergic and glutamatergic antagonists, and we postulated that it was controlled by CR cells. We now investigate the correlated activity of embryonic and postnatal layer 1 in wild-type and reeler mice, mutant in the production of reelin. We find that mouse layer 1 also sustains patterned spontaneous activity and that CR cells participate in correlated networks. These networks are present in embryonic marginal zone and are blocked by GABAergic and glutamatergic antagonists. Surprisingly, network activity in reeler mice displays similar characteristics and pharmacological profile as in wild-type mice, although small differences are detected. Our results demonstrate that the embryonic marginal zone has correlated spontaneous activity that could serve as the scaffold for the development of intracortical connections. Our data also suggest that reelin does not have a major impact in the development of specific synaptic circuits in layer 1.

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PubMed1999

Developmental regulation of spine motility in the mammalian central nervous system.

The function of dendritic spines, postsynaptic sites of excitatory input in the mammalian central nervous system (CNS), is still not well understood. Although changes in spine morphology may mediate synaptic plasticity, the extent of basal spine motility and its regulation and function remains controversial. We investigated spine motility in three principal neurons of the mouse CNS: cerebellar Purkinje cells, and cortical and hippocampal pyramidal neurons. Motility was assayed with time-lapse imaging by using two-photon microscopy of green fluorescent protein-labeled neurons in acute and cultured slices. In all three cell types, dendritic protrusions (filopodia and spines) were highly dynamic, exhibiting a diversity of morphological rearrangements over short (<1-min) time courses. The incidence of spine motility declined during postnatal maturation, but dynamic changes were still apparent in many spines in late-postnatal neurons. Although blockade or induction of neuronal activity did not affect spine motility, disruption of actin polymerization did. We hypothesize that this basal motility of dendritic protrusions is intrinsic to the neuron and underlies the heightened plasticity found in developing CNS.

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