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Cajal's description of the neuron as the structural and functional unit of the nervous system formed the basis of much subsequent neuroscientific research. In this Timeline article, Yuste considers the contribution of the 'neuron doctrine' to neuroscience and discusses the recent transition in paradigm from the single neuron to neural networks. For over a century, the neuron doctrine — which states that the neuron is the structural and functional unit of the nervous system — has provided a conceptual foundation for neuroscience. This viewpoint reflects its origins in a time when the use of single-neuron anatomical and physiological techniques was prominent. However, newer multineuronal recording methods have revealed that ensembles of neurons, rather than individual cells, can form physiological units and generate emergent functional properties and states. As a new paradigm for neuroscience, neural network models have the potential to incorporate knowledge acquired with single-neuron approaches to help us understand how emergent functional states generate behaviour, cognition and mental disease.

The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation–dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations.

To better understand the input-output computations of neuronal populations, we developed ArcLight-ST, a genetically-encoded voltage indicator, to specifically measure subthreshold membrane potentials. We combined two-photon imaging of voltage and calcium, and successfully discriminated subthreshold inputs and spikes with cellular resolution in vivo. We demonstrate the utility of the method by mapping epileptic seizures progression through cortical circuits, revealing divergent sub- and suprathreshold dynamics within compartmentalized epileptic micronetworks. Two-photon, two-color imaging of calcium and voltage enables mapping of inputs and outputs in neuronal populations in living animals. The authors present a geneticallyencoded voltage indicator to specifically measure subthreshold membrane potentials. They combine two-photon imaging of voltage and calcium to map epileptic seizures progression through cortical circuits.

The study of electronic properties of materials at the nanoscale has unveiled physical laws and generated materials such as nanoparticles, quantum dots, nanodiamonds, nanoelectrodes, and nanoprobes. Independently, large-scale public and private neuroscience programs have been launched to develop methods to measure and manipulate neural circuits in living animals and humans. Here, we review an upcoming field, NanoNeuro, defined as the intersection of nanoscience and neuroscience, that aims to develop nanoscale methods to record and stimulate neuronal activity. Because of their unique physical properties, nanomaterials have intrinsic advantages as biosensors and actuators, and they may be applicable to humans without the need for genetic modifications. Thus, nanoscience could make major methodological contributions to the future of neuroscience and, more generally, to biomedical sciences.This Perspective discusses how neuroscience can benefit from harnessing nanoscience-based tools for manipulating and recording neuronal activity.

The simultaneous imaging and manipulating of neural activity could enable the functional dissection of neural circuits. Here we have combined two-photon optogenetics with simultaneous volumetric two-photon calcium imaging to measure and manipulate neural activity in mouse neocortex in vivo in three-dimensions (3D) with cellular resolution. Using a hybrid holographic approach, we simultaneously photostimulate more than 80 neurons over 150 μm in depth in layer 2/3 of the mouse visual cortex, while simultaneously imaging the activity of the surrounding neurons. We validate the usefulness of the method by photoactivating in 3D selected groups of interneurons, suppressing the response of nearby pyramidal neurons to visual stimuli in awake animals. Our all-optical approach could be used as a general platform to read and write neuronal activity. Modern microscopy provides a window into the brain. The first light microscopes were able to magnify cells only in thin slices of tissue. By contrast, today’s light microscopes can image cells below the surface of the brain of a living animal. Even so, this remains challenging for several reasons. One is that the brain is three-dimensional. Another is that brain tissue scatters light. Trying to view neurons deep within the brain is a little like trying to view them through a glass of milk. Most of the light scatters on its way through the tissue with the result that little of the light reaches the target neurons. Yang et al. have now tackled these challenges using a technique called holography. Holography produces 3D images of objects by splitting a beam of light and then recombining the beams in a specific way. Yang et al. applied this technique to an infrared laser beam, opting for infrared because it scatters much less in brain tissue than visible light. Directing each of the infrared beams to a different neuron can produce 3D images of multiple cells within the brain’s outer layer, the cortex, all at the same time. The holographic infrared microscope can be used alongside two techniques called optogenetics and calcium imaging, in which light-sensitive proteins are inserted into neurons. Depending on the proteins introduced, shining light onto the neurons will either change their activity, or cause them to fluoresce whenever they are active. Just as a computer can both “read” and “write” data, the holographic microscope can thus read out existing neuronal activity or write new patterns of activity. By combining these techniques, Yang et al. were able to stimulate more than 80 neurons at the same time – and meanwhile visualize the activity of the surrounding neurons – at multiple depths within the mouse cortex. This new microscopy technique, while a clear advance over existing methods, still cannot image and control neurons throughout the entire cortex. The next goal is to further extend this method across multiple brain areas and manipulate the activity of any subset of neurons at will. Neuroscientists will greatly benefit from the ability to image and alter the activity of living neural circuits in 3D. In the future, clinicians may be able to use this technique to treat brain disorders by adjusting the activity of abnormal neural circuits.

Neuronal ensembles, coactive groups of neurons found in spontaneous and evoked cortical activity, are causally related to memories and perception, but it is still unknown how stable or flexible they are over time. We used two-photon multiplane calcium imaging to track over weeks the activity of the same pyramidal neurons in layer 2/3 of the visual cortex from awake mice and recorded their spontaneous and visually evoked responses. Less than half of the neurons remained active across any two imaging sessions. These stable neurons formed ensembles that lasted weeks, but some ensembles were also transient and appeared only in one single session. Stable ensembles preserved most of their neurons for up to 46 days, our longest imaged period, and these ‘core’ cells had stronger functional connectivity. Our results demonstrate that neuronal ensembles can last for weeks and could, in principle, serve as a substrate for long-lasting representation of perceptual states or memories.

Synaptic connectivity defines groups of neurons that engage in correlated activity during specific functional tasks. These co-active groups of neurons form ensembles, the operational units involved in, for example, sensory perception, motor coordination and memory (then called an engram ). Traditionally, ensemble formation has been thought to occur via strengthening of synaptic connections via long-term potentiation (LTP) as a plasticity mechanism. This synaptic theory of memory arises from the learning rules formulated by Hebb and is consistent with many experimental observations. Here, we propose, as an alternative, that the intrinsic excitability of neurons and its plasticity constitute a second, non-synaptic mechanism that could be important for the initial formation of ensembles. Indeed, enhanced neural excitability is widely observed in multiple brain areas subsequent to behavioral learning. In cortical structures and the amygdala, excitability changes are often reported as transient, even though they can last tens of minutes to a few days. Perhaps it is for this reason that they have been traditionally considered as modulatory, merely supporting ensemble formation by facilitating LTP induction, without further involvement in memory function (memory allocation hypothesis). We here suggest−based on two lines of evidence—that beyond modulating LTP allocation, enhanced excitability plays a more fundamental role in learning. First, enhanced excitability constitutes a signature of active ensembles and, due to it, subthreshold synaptic connections become suprathreshold in the absence of synaptic plasticity ( iceberg model ). Second, enhanced excitability promotes the propagation of dendritic potentials toward the soma and allows for enhanced coupling of EPSP amplitude (LTP) to the spike output (and thus ensemble participation). This permissive gate model describes a need for permanently increased excitability, which seems at odds with its traditional consideration as a short-lived mechanism. We propose that longer modifications in excitability are made possible by a low threshold for intrinsic plasticity induction, suggesting that excitability might be on/off-modulated at short intervals. Consistent with this, in cerebellar Purkinje cells, excitability lasts days to weeks, which shows that in some circuits the duration of the phenomenon is not a limiting factor in the first place. In our model, synaptic plasticity defines the information content received by neurons through the connectivity network that they are embedded in. However, the plasticity of cell-autonomous excitability could dynamically regulate the ensemble participation of individual neurons as well as the overall activity state of an ensemble.

Neuronal ensembles are coactive groups of neurons that may represent building blocks of cortical circuits. These ensembles could be formed by Hebbian plasticity, whereby synapses between coactive neurons are strengthened. Here we report that repetitive activation with two-photon optogenetics of neuronal populations from the visual cortex of awake mice builds neuronal ensembles that recur spontaneously after being imprinted and do not disrupt preexisting ones. Moreover, imprinted ensembles can be recalled by single-cell stimulation and remain coactive on consecutive days. Our results demonstrate the persistent reconfiguration of cortical circuits by two-photon optogenetics into neuronal ensembles that can perform pattern completion.

Large brain tissue sections are imaged with nanoscale resolution using expansion and lattice light sheet microscopy.

Most excitatory inputs in the mammalian brain are made on dendritic spines, rather than on dendritic shafts. Spines compartmentalize calcium, and this biochemical isolation can underlie input-specific synaptic plasticity, providing a raison d’etre for spines. However, recent results indicate that the spine can experience a membrane potential different from that in the parent dendrite, as though the spine neck electrically isolated the spine. Here we use two-photon calcium imaging of mouse neocortical pyramidal neurons to analyze the correlation between the morphologies of spines activated under minimal synaptic stimulation and the excitatory postsynaptic potentials they generate. We find that excitatory postsynaptic potential amplitudes are inversely correlated with spine neck lengths. Furthermore, a spike timing-dependent plasticity protocol, in which two-photon glutamate uncaging over a spine is paired with postsynaptic spikes, produces rapid shrinkage of the spine neck and concomitant increases in the amplitude of the evoked spine potentials. Using numerical simulations, we explore the parameter regimes for the spine neck resistance and synaptic conductance changes necessary to explain our observations. Our data, directly correlating synaptic and morphological plasticity, imply that long-necked spines have small or negligible somatic voltage contributions, but that, upon synaptic stimulation paired with postsynaptic activity, they can shorten their necks and increase synaptic efficacy, thus changing the input/output gain of pyramidal neurons.