Rafael Yuste

Fluorescence microscopy today

Fluorescence microscopy has undergone a renaissance in the last decade. The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues. The impact of these and other new imaging methods in biophysics, neuroscience, and developmental and cell biology has been remarkable. Further advances in fluorophore design, molecular biological tools and nonlinear and hyper-resolution microscopies are poised to profoundly transform many fields of biological research.

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

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.

Genesis of dendritic spines: insights from ultrastructural and imaging studies

Dendritic spines are small protrusions from many types of neuron, which receive most of the excitatory inputs to the cell. Spines are thought to have important roles in neural information processing and plasticity, yet we still have a poor understanding of how they emerge during development. Here, we review the developmental generation of dendritic spines, covering recent live imaging experiments and older ultrastructural data. We address the potential role of dendritic filopodia in spine development and recent findings of spinogenesis in adult animals, and conclude by discussing three potential models of spinogenesis.

Dendritic Integration in Mammalian Neurons, a Century after Cajal

We thank A. Borst, B. W. Connors, T. Freund, S. J. Redman, W. N. Ross, T. J. Sejnowski, and R. D. Traub for sharing their results before publication, A. Borst, S. Cash, B. W. Connors, W. Denk, S. R. Golob, R. Llinas, W. Regehr, P. A. Rhodes, I. Segev, and K. Svoboda for helpful comments, and A. Gelperin, J. Lichtman, Z. Mainem, V. Murthy, G. M. Shepherd, L. Trussell, and R. O. L. Wong for their help. R. Y. was supported by the Office of Naval Research.

Extensive dye coupling between rat neocortical neurons during the period of circuit formation

A low molecular weight intracellular tracer, Neurobiotin, was injected into single neurons in living slices of rat neocortex made at postnatal days 5-18. Between days 5 and 12, 66% of single-neuron injections labeled clusters of up to 80 neurons surrounding the injected cell. Coupling between neurons occurred primarily through dendrites. Injections done in the presence of halothane, a gap junction blocker, abolished the spread of tracer to surrounding neurons, implying that gap junctions mediate coupling. Injections done after day 16 resulted in little or no dye coupling. We conclude that transient local coupling via gap junctions in developing cortex may provide a pathway for communicating intercellular signals, including subthreshold electrical activity, and thereby enable temporal coordination of local neuronal ensembles during circuit formation.

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.

Dense Inhibitory Connectivity in Neocortex

The connectivity diagram of neocortical circuits is still unknown, and there are conflicting data as to whether cortical neurons are wired specifically or not. To investigate the basic structure of cortical microcircuits, we use a two-photon photostimulation technique that enables the systematic mapping of synaptic connections with single-cell resolution. We map the inhibitory connectivity between upper layers somatostatin-positive GABAergic interneurons and pyramidal cells in mouse frontal cortex. Most, and sometimes all, inhibitory neurons are locally connected to every sampled pyramidal cell. This dense inhibitory connectivity is found at both young and mature developmental ages. Inhibitory innervation of neighboring pyramidal cells is similar, regardless of whether they are connected among themselves or not. We conclude that local inhibitory connectivity is promiscuous, does not form subnetworks, and can approach the theoretical limit of a completely connected synaptic matrix.

The Brain Activity Map Project and the Challenge of Functional Connectomics

The function of neural circuits is an emergent property that arises from the coordinated activity of large numbers of neurons. To capture this, we propose launching a large-scale, international public effort, the Brain Activity Map Project, aimed at reconstructing the full record of neural activity across complete neural circuits. This technological challenge could prove to be an invaluable step toward understanding fundamental and pathological brain processes.

Internal Dynamics Determine the Cortical Response to Thalamic Stimulation

Although spontaneous activity occurs throughout the neocortex, its relation to the activity produced by external or sensory inputs remains unclear. To address this, we used calcium imaging of mouse thalamocortical slices to reconstruct, with single-cell resolution, the spatiotemporal dynamics of activity of layer 4 in the presence or absence of thalamic stimulation. We found spontaneous neuronal coactivations corresponded to intracellular UP states. Thalamic stimulation of sufficient frequency (>10 Hz) triggered cortical activity, and UP states, indistinguishable from those arising spontaneously. Moreover, neurons were activated in identical and precise spatiotemporal patterns in thalamically triggered and spontaneous events. The similarities between cortical activations indicate that intracortical connectivity plays the dominant role in the cortical response to thalamic inputs. Our data demonstrate that precise spatiotemporal activity patterns can be triggered by thalamic inputs and indicate that the thalamus serves to release intrinsic cortical dynamics.