Biomedical subjects

Rafael Yuste

Publications and source records attributed to Rafael Yuste.

At least 19 recordsLinked to original sources

PubMed2003

Activity-regulated dynamic behavior of early dendritic protrusions: evidence for different types of dendritic filopodia.

Dendritic filopodia are long and thin protrusions that occur predominantly during early development of the mammalian CNS. The function of dendritic filopodia is unknown, but they could serve to form early synapses, to generate spines, or to regulate dendritic branching and growth. We used two-photon imaging to characterize the motile behavior of dendritic protrusions during early postnatal development (P2-P12) in pyramidal neurons from acute slices of mouse neocortex. Dendritic protrusions in immature neurons are highly dynamic, and this motility is actin based. Motility and turnover of these early protrusions decreases throughout development, mirroring an increase in their average lifetime and density. Interestingly, density, motility, and length of filopodia are greater in dendritic growth cones than in dendritic shafts. These growth cones disappear after P5. Blocking synaptic transmission globally using TTX or calcium-free solutions led to a 40-120% increase in the density and length of dendritic filopodia in shafts but not in growth cones. Moreover, blocking ionotropic glutamate receptors resulted in an approximately 35% decrease in the density and turnover of shaft filopodia, whereas focal glutamate application led to a 75% increase in the length of shaft filopodia, but neither manipulation affected growth cone filopodia. Our results support the existence of two populations of filopodia, in growth cones and shafts, which are differentially regulated by neuronal activity. We propose that filopodia in dendritic growth cones are involved in dendritic growth and branching in an activity-independent manner, whereas shaft filopodia are responsible for activity-dependent synaptogenesis and, in some cases, may become dendritic spines.

Animals↗

PubMed2003

Quantitative morphologic classification of layer 5 neurons from mouse primary visual cortex.

The understanding of any neural circuit requires the identification and characterization of all its components. Morphologic classifications of neurons are, therefore, of central importance to neuroscience. We use a quantitative method to classify neurons from layer 5 of mouse primary visual cortex, based on multidimensional clustering. To reconstruct neurons, we used Golgi impregnations and biocytin injections, as well as DiOlistics, a novel technique of labeling neurons with lipophilic dyes. We performed computerized 3-D reconstructions of 158 layer 5 cells to measure a series of morphologic variables. Principal component analysis and cluster analysis were used for the classification of cell types. Five major classes of cells were found: group 1 includes large pyramidal neurons with apical dendrites that reach layer 1 with an apical tuft; group 2 consists of short pyramidal neurons and large multipolar cells with "polarized" dendritic trees; group 3 is composed of less extensive pyramidal neurons; group 4 includes small cells; and group 5 includes another set of short pyramidal neurons in addition to "atypically oriented" cells. Our sample included a relatively homogeneous group of 27 neurons that project to the superior colliculus, which clustered mainly in group 1, thus supporting the validity of the classification. Cluster analysis of neuronal morphologies provides an objective method to quantitatively define different neuronal phenotypes and may serve as a basis for describing neocortical circuits.

Animals↗

PubMed2003

Ca2+ imaging of mouse neocortical interneurone dendrites: Ia-type K+ channels control action potential backpropagation.

GABAergic interneurones are essential in cortical processing, yet the functional properties of their dendrites are still poorly understood. In this first study, we combined two-photon calcium imaging with whole-cell recording and anatomical reconstructions to examine the calcium dynamics during action potential (AP) backpropagation in three types of V1 supragranular interneurones: parvalbumin-positive fast spikers (FS), calretinin-positive irregular spikers (IS), and adapting cells (AD). Somatically generated APs actively backpropagated into the dendritic tree and evoked instantaneous calcium accumulations. Although voltage-gated calcium channels were expressed throughout the dendritic arbor, calcium signals during backpropagation of both single APs and AP trains were restricted to proximal dendrites. This spatial control of AP backpropagation was mediated by Ia-type potassium currents and could be mitigated by by previous synaptic activity. Further, we observed supralinear summation of calcium signals in synaptically activated dendritic compartments. Together, these findings indicate that in interneurons, dendritic AP propagation is synaptically regulated. We propose that interneurones have a perisomatic and a distal dendritic functional compartment, with different integrative functions.

Action Potentials↗

PubMed2003

Ca2+ imaging of mouse neocortical interneurone dendrites: contribution of Ca2+-permeable AMPA and NMDA receptors to subthreshold Ca2+dynamics.

In this second study, we have combined two-photon calcium imaging with whole-cell recording and anatomic reconstructions to directly characterize synaptically evoked calcium signals in three types of mouse V1 supragranular interneurones: parvalbumin-positive fast spikers (FS), calretinin-positive irregular spikers (IS), and adapting cells (AD). We observed that subthreshold synaptic activation evoked calcium signals locally restricted to individual dendritic compartments. These signals were mediated by NMDA receptors (NMDARs) in AD and IS cells, whereas in FS cells, calcium-permeable AMPA receptors (CP-AMPARs) provided an additional and kinetically distinct influx. Furthermore, even a single, subthreshold synaptic activation evoked a larger dendritic calcium influx than backpropagating action potentials. Our results demonstrate that NMDARs dominate subthreshold calcium dynamics in interneurones and reveal the functional contribution of CP-AMPARs to a specific subclass of cortical interneurone. These data highlight different strategies in dendritic signal processing by distinct classes of interneurones.

Action Potentials↗

PubMed2003

Bidirectional regulation of hippocampal mossy fiber filopodial motility by kainate receptors: a two-step model of synaptogenesis.

The rapid motility of axonal filopodia and dendritic spines is prevalent throughout the developing CNS, although the function of this motility remains controversial. Using two-photon microscopy, we imaged hippocampal mossy fiber axons in slice cultures and discovered that filopodial extensions are highly motile. Axonal filopodial motility is actin based and is downregulated with development, although it remains in mature cultures. This motility is correlated with free extracellular space yet is inversely correlated with contact with postsynaptic targets, indicating a potential role in synaptogenesis. Filopodial motility is differentially regulated by kainate receptors: synaptic stimulation of kainate receptors enhances motility in younger slices, but it inhibits it in mature slices. We propose that neuronal activity controls filopodial motility in a developmentally regulated manner, in order to establish synaptic contacts in a two-step process. A two-step model of synaptogenesis can also explain the opposite effects of neuronal activity on the motility of dendritic protrusions.

Actin Cytoskeleton↗

PubMed2003

Attractor dynamics of network UP states in the neocortex.

The cerebral cortex receives input from lower brain regions, and its function is traditionally considered to be processing that input through successive stages to reach an appropriate output. However, the cortical circuit contains many interconnections, including those feeding back from higher centres, and is continuously active even in the absence of sensory inputs. Such spontaneous firing has a structure that reflects the coordinated activity of specific groups of neurons. Moreover, the membrane potential of cortical neurons fluctuates spontaneously between a resting (DOWN) and a depolarized (UP) state, which may also be coordinated. The elevated firing rate in the UP state follows sensory stimulation and provides a substrate for persistent activity, a network state that might mediate working memory. Using two-photon calcium imaging, we reconstructed the dynamics of spontaneous activity of up to 1,400 neurons in slices of mouse visual cortex. Here we report the occurrence of synchronized UP state transitions ('cortical flashes') that occur in spatially organized ensembles involving small numbers of neurons. Because of their stereotyped spatiotemporal dynamics, we conclude that network UP states are circuit attractors--emergent features of feedback neural networks that could implement memory states or solutions to computational problems.

Animals↗

PubMed2003

Systematic regulation of spine sizes and densities in pyramidal neurons.

Dendritic spines receive most excitatory inputs in the CNS. Recent evidence has demonstrated that the spine head volume is linearly correlated with the readily releasable pool of neurotransmitter and the PSD size. These correlations can be used to functionally interpret spine morphology. Using Golgi impregnations and light microscopy, we reconstructed 23000 spines from pyramidal neurons in layers 2/3, 4, 5 and 6 of mouse primary visual cortex and CA1 hippocampal region and measured their spine head diameters and densities. Spine head diameters and densities are variable within and across cells, although they are similar between apical and basal dendrites. When compared to other regions, layer 5 neurons have larger spine heads and CA1 neurons higher spine densities. Interestingly, we detect a correlation between spine head diameter and interspine distance within and across cells, whereby larger spines are spaced further away from each other than smaller spines. Finally, in CA1 neurons, spine head diameters are larger, and spine density lower, in distal apical dendrites (>200 microm from soma) compared to proximal regions. These results reveal that spine morphologies and densities, and therefore synaptic properties, are jointly modulated with respect to cortical region, laminar position, and, in some cases, even the position of the spine along the dendritic tree. Individual neurons also appear to regulate their apical and basal spine densities and morphologies in concert. Our data provide evidence for a homeostatic control of excitatory synaptic strength.

Animals↗

PubMed2003

A two-photon and second-harmonic microscope.

Two-photon microscopy has revolutionized life sciences by enabling long-term imaging of living preparations in highly scattering tissue while minimizing photodamage. At the same time, commercial two-photon microscopes are expensive and this has prevented the widespread application of this technique to the biological community. As an alternative to commercial systems, we provide an update of our efforts designing custom-built two-photon instruments by modifying the Olympus FluoView laser scanning confocal microscope. With the newer version of our instrument we modulate the intensity of the laser beam in arbitrary spatiotemporal patterns using a Pockels cell and software control over the scanning. We can also perform simultaneous optical imaging and optical stimulation experiments and combine them with second harmonic generation measurements.

Animals↗

PubMed2003

Morphological and physiological characterization of layer VI corticofugal neurons of mouse primary visual cortex.

Layer VI is the origin of the massive feedback connection from the cortex to the thalamus, yet its complement of cell types and their connections is poorly understood. The physiological and morphological properties of corticofugal neurons of layer VI of mouse primary visual cortex were investigated in slices loaded with the Ca(2+) indicator fura-2AM. To identify corticofugal neurons, electrical stimulation of the white matter (WM) was done in conjunction with calcium imaging to detect neurons that responded with changes in intracellular Ca(2+) concentrations in response to the stimulation. Subsequent whole cell recordings confirmed that they discharged antidromic action potentials after WM stimulation. Antidromically activated neurons were more excitable and had different spiking properties than neighboring nonantidromic neurons, although both groups had similar input resistances. Furthermore, antidromic neurons possessed narrower action potentials and smaller afterhyperpolarizations. Additionally, three-dimensional reconstructions indicated that antidromically activated neurons had a distinct morphology with longer apical dendrites and fewer nonprimary dendrites than nonantidromic cells. To identify the antidromic neurons, rhodamine microspheres were injected into the dorsal lateral geniculate nucleus of the thalamus and allowed to retrogradely transport back to the somata of the layer VI cortico-geniculate neurons. Physiological and anatomical analysis indicated that most antidromic neurons were likely to be cortico-geniculate neurons. Our results show that cortico-thalamic neurons represent a specific functional and morphological class of layer VI neurons.

Action Potentials↗

PubMed2003

BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl- co-transporter KCC2.

Spontaneous neural activity is a basic property of the developing brain, which regulates key developmental processes, including migration, neural differentiation and formation and refinement of connections. The mechanisms regulating spontaneous activity are not known. By using transgenic embryos that overexpress BDNF under the control of the nestin promoter, we show here that BDNF controls the emergence and robustness of spontaneous activity in embryonic hippocampal slices. Further, BDNF dramatically increases spontaneous co-active network activity, which is believed to synchronize gene expression and synaptogenesis in vast numbers of neurons. In fact, BDNF raises the spontaneous activity of E18 hippocampal neurons to levels that are typical of postnatal slices. We also show that BDNF overexpression increases the number of synapses at much earlier stages (E18) than those reported previously. Most of these synapses were GABAergic, and GABAergic interneurons showed hypertrophy and a 3-fold increase in GAD expression. Interestingly, whereas BDNF does not alter the expression of GABA and glutamate ionotropic receptors, it does raise the expression of the recently cloned K(+)/Cl(-) KCC2 co-transporter, which is responsible for the conversion of GABA responses from depolarizing to inhibitory, through the control of the Cl(-) potential. Together, results indicate that both the presynaptic and postsynaptic machineries of GABAergic circuits may be essential targets of BDNF actions to control spontaneous activity. The data indicate that BDNF is a potent regulator of spontaneous activity and co-active networks, which is a new level of regulation of neurotrophins. Given that BDNF itself is regulated by neuronal activity, we suggest that BDNF acts as a homeostatic factor controlling the emergence, complexity and networking properties of spontaneous networks.

Animals↗

PubMed2002

Thalamocortical bursts trigger recurrent activity in neocortical networks: layer 4 as a frequency-dependent gate.

Sensory information reaches the cortex via thalamocortical (TC) synapses in layer 4. Thalamic relay neurons that mediate information flow to cortex operate in two distinct modes, tonic and burst firing. Burst firing has been implicated in enhancing reliability of information flow between individual neurons. However, little is known about how local networks of neocortical neurons respond to different temporal patterns of TC activity. We studied cortical activity patterns evoked by stimulating TC afferents at different frequencies, using a combination of electrophysiology and calcium imaging in TC slices that allowed for the reconstruction of spatiotemporal activity with single-cell resolution. Stimulation of TC axons at low frequencies triggered action potentials in only a small number of layer 4 neurons. In contrast, brief high-frequency stimulus trains triggered widespread recurrent activity in populations of neurons in layer 4 and then spread into adjacent layers 2/3 and 5. Recurrent activity had a clear threshold, typically lasted 300 msec, and could be evoked repetitively at frequencies up to 0.5 Hz. Moreover, the spatial extent of recurrent activity was controlled by the TC pattern of activity. Recurrent activity triggered within the highly interconnected networks of layer 4 might act to selectively amplify and redistribute transient high-frequency TC inputs, filter out low-frequency inputs, and temporarily preserve a record of past sensory activity.

Action Potentials↗

PubMed2002

Spine motility. Phenomenology, mechanisms, and function.

Throughout the history of neuroscience, dendritic spines have been considered stable structures, but in recent years, imaging techniques have revealed that spines are constantly changing shape. Spine motility is difficult to categorize, has different forms, and possibly even represents multiple phenomena. It is influenced by synaptic transmission, intracellular calcium, and a multitude of ions and other molecules. An actin-based cascade mediates this phenomenon, and while the precise signaling pathways are still unclear, the Rho family of GTPases could well be a "common denominator" controlling spine morphology. One role of spine motility might be to enable a searching function during synaptogenesis, allowing for more efficacious neuronal connectivity in the neuronal thicket. This idea revisits concepts originally formulated by Cajal, who proposed over a hundred years ago that spines might help to increase and modify synaptic connections.

Animals↗

PubMed2002

Multiphoton stimulation of neurons.

We pulsed the activation of neurons using a femtosecond laser. Pyramidal neurons are depolarized and fire action potentials when high intensity mode-locked infrared light irradiates somatic membranes and axon initial segments. This depolarization is reversible, does not occur with CW laser light, and appears to be due to multiphoton excitation. We describe two regimes of multiphoton optical stimulation. Low intensity, long duration laser irradiation produces a sustained depolarization, insensitive to sodium channel blockers yet sensitive to antioxidants. On the other hand, high intensity, short duration irradiation can induce fast depolarizations, which appear due to different mechanism. The combination of multiphoton stimulation and optical probing could enable systematic analysis of circuits.

Action Potentials↗

PubMed2002

Calcium dynamics of spines depend on their dendritic location.

Dendritic spines are morphologically and functionally heterogeneous. To understand this diversity, we use two-photon imaging of layer 5 neocortical pyramidal cells and measure action potential-evoked [Ca(2+)]i transients in spines. Spine calcium kinetics are controlled by (i) the diameter of the parent dendrite, (ii) the length of the spine neck, and (iii) the strength of spine calcium pumps. These factors produce different calcium dynamics in spines from basal, proximal apical, and distal apical dendrites, differences that are more pronounced without exogenous buffers. In proximal and distal apical dendrites, different calcium dynamics correlate with different susceptibility to synaptic depression, and modifying calcium kinetics in spines changes the expression of long-term depression. Thus, the spine location apparently determines its calcium dynamics and synaptic plasticity. Our results highlight the precision in design of neocortical neurons.

Animals↗

PubMed2002

Calcium oscillations in neocortical astrocytes under epileptiform conditions.

Morphological and functional alterations in astrocytic glia are often found in epileptic syndromes, although the exact role of astrocytes in epilepsy is poorly understood. During calcium imaging of epileptiform events in juvenile neocortical slices we previously discovered cells with spontaneous oscillations in their intracellular free calcium concentration ([Ca(2+)](i)). We have now characterized these oscillations using two in vitro models of epilepsy and find that they are produced by astrocytes. Astrocytic oscillations are widespread throughout the imaged territories, are remarkably regular and have long periods, averaging 100 s, which become shorter during development. Astrocytic oscillations are uncorrelated among themselves and with epileptiform events, are blocked by internal release antagonists and are stimulated by caffeine. Astrocytic calcium oscillations could mediate reactive astrogliosis, contribute to the pathogenesis of chronic epileptic syndromes, and be used as a diagnostic test for epileptic tissue.

Animals↗

PubMed2002

A problem with Hebb and local spikes.

Although our understanding of the cellular properties of mammalian neurons is increasing rapidly, the computational function of their elaborate dendritic trees is still mysterious. In recent years, experiments have shown that, in pyramidal cells, individual dendritic compartments sustain local excitation spikes.. These dendrites also support Hebbian synaptic plasticity, which depends on the precise temporal relationship between pre- and postsynaptic spikes. In this review we discuss what we consider to be a problem with Hebbian (i.e., spike-timing-dependent) plasticity. We argue that most of the spikes that occur in dendrites are not back-propagating action potentials but but local spikes, and that Hebbian plasticity caused by local spikes can undermine the functional integrity of the geometrically complex dendritic tree. We propose that the inverted Hebbian plasticity of synapses involved in local spikes, and/or local dendritic homeostatic plasticity, could prevent an unbalanced distribution of synaptic weights on the dendritic tree.

Animals↗

PubMed2002

Stimulating neurons with light.

Recent technological advances have enabled the use of different optical methods to activate neurons, including 'caged' glutamate, photoactivation of genetically engineered cascades, and direct two-photon excitation. The ability to use light as a stimulation tool provides, in principle, a non-invasive method for the temporally and spatially precise activation of any neuron or any part of a neuron. When combined with two-photon excitation, excellent spatial control can be achieved even in complex and highly scattering preparations, such as living nervous tissue. Different methods that have been developed in the last several decades have been used to probe neuronal sensitivity, mimic synaptic input, and elucidate patterns of neural connectivity.

Animals↗

PubMed2002

Analysis of multineuronal activation patterns from calcium-imaging experiments in brain slices.

The patterns of cellular organization and intercellular communication in excitable tissues are extraordinarily complex. This article reviews a new set of tools for the imaging and statistical analysis of multicellular circuits in real time, and describes the findings from analysis of neuronal networks from slices of rodent neocortex loaded with fluorescent calcium indicators. Spontaneous activation of multineuronal networks was found to be generated by both synaptic and intrinsic mechanisms, and these cortical microcircuits have a precise spatial organization. In general, these techniques and methods of analysis, which can detect patterns of activity with single-cell and millisecond resolution, are applicable to the study of calcium and electrical signaling in cell populations of excitable tissues such as the heart, skeletal muscle, and the nervous system.

Animals↗