Controlling Visually Guided Behavior by Holographic Recalling of Cortical Ensembles.

Neurons in cortical circuits are often coactivated as ensembles, yet it is unclear whether ensembles play a functional role in behavior. Some ensemble neurons have pattern completion properties, triggering the entire ensemble when activated. Using two-photon holographic optogenetics in mouse primary visual cortex, we tested whether recalling ensembles by activating pattern completion neurons alters behavioral performance in a visual task. Disruption of behaviorally relevant ensembles by activation of non-selective neurons decreased performance, whereas activation of only two pattern completion neurons from behaviorally relevant ensembles improved performance, by reliably recalling the whole ensemble. Also, inappropriate behavioral choices were evoked by the mistaken activation of behaviorally relevant ensembles. Finally, in absence of visual stimuli, optogenetic activation of two pattern completion neurons could trigger behaviorally relevant ensembles and correct behavioral responses. Our results demonstrate a causal role of neuronal ensembles in a visually guided behavior and suggest that ensembles implement internal representations of perceptual states.

Toward a Global BRAIN Initiative.

Neuroscience is entering a collaborative era in which powerful new technologies, generated by large scientific projects in many countries, will have a dramatic impact on science, medicine, and society. Coordinating these international initiatives and ensuring broad distribution of novel technologies and open accessibility of the generated data will multiply their value, while tapping creativity and expertise from every source.

On the Necessity of Ethical Guidelines for Novel Neurotechnologies.

Because novel neurotechnologies may alter human identity and society in profound ways, we advocate for the early integration of ethics into neurotechnology. We recommend developing and adopting a set of guidelines, like the Belmont Report on human subject research, as a framework for development and use of brain-related technologies.

A complete biomechanical model of Hydra contractile behaviors, from neural drive to muscle to movement.

How does neural activity drive muscles to produce behavior? The recent development of genetic lines in Hydra that allow complete calcium imaging of both neuronal and muscle activity, as well as systematic machine learning quantification of behaviors, makes this small cnidarian an ideal model system to understand and model the complete transformation from neural firing to body movements. To achieve this, we have built a neuromechanical model of Hydra's fluid-filled hydrostatic skeleton, showing how drive by neuronal activity activates distinct patterns of muscle activity and body column biomechanics. Our model is based on experimental measurements of neuronal and muscle activity and assumes gap junctional coupling among muscle cells and calcium-dependent force generation by muscles. With these assumptions, we can robustly reproduce a basic set of Hydra's behaviors. We can further explain puzzling experimental observations, including the dual timescale kinetics observed in muscle activation and the engagement of ectodermal and endodermal muscles in different behaviors. This work delineates the spatiotemporal control space of Hydra movement and can serve as a template for future efforts to systematically decipher the transformations in the neural basis of behavior.

RuBi-Ruxolitinib: A Photoreleasable Antitumor JAK Inhibitor.

We describe the synthesis and biological testing of ruthenium-bipyridine ruxolitinib (RuBiRuxo), a photoreleasable form of ruxolitinib, a JAK inhibitor used as an antitumoral agent in cutaneous T-cell lymphomas (CTCL). This novel caged compound is synthesized efficiently, is stable in aqueous solution at room temperature, and is photoreleased rapidly by visible light. Irradiation of RuBiRuxo reduces cell proliferation and induces apoptosis in a light- and time-dependent manner in a CTCL cell line. This effect is specific and is mediated by a decreased phosphorylation of STAT proteins. Our results demonstrate the potential of ruthenium-based photocompounds and light-based therapeutic approaches for the potential treatment of cutaneous lymphomas and other pathologies.

Time for NanoNeuro.

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.

Author Correction: The new nanophysiology: regulation of ionic flow in neuronal subcompartments.

In this article, the origin of some of the presented modelling information has been stated and more details have been provided to understand how the curve in Box 2 and the electrodiffusion-related curves in Figure 3 were generated.

Publisher Correction: The new nanophysiology: regulation of ionic flow in neuronal subcompartments.

In parts b and c of Figure 3, the y axes were incorrectly labelled 'Concentration (µM)'. They should have been labelled 'Concentration (mM)'. The corrected figure is shown below.

Cortical ensembles selective for context.

Neural processing of sensory information is strongly influenced by context. For instance, cortical responses are reduced to predictable stimuli, while responses are increased to novel stimuli that deviate from contextual regularities. Such bidirectional modulation based on preceding sensory context is likely a critical component or manifestation of attention, learning, and behavior, yet how it arises in cortical circuits remains unclear. Using volumetric two-photon calcium imaging and local field potentials in primary visual cortex (V1) from awake mice presented with visual "oddball" paradigms, we identify both reductions and augmentations of stimulus-evoked responses depending, on whether the stimulus was redundant or deviant, respectively. Interestingly, deviance-augmented responses were limited to a specific subset of neurons mostly in supragranular layers. These deviance-detecting cells were spatially intermixed with other visually responsive neurons and were functionally correlated, forming a neuronal ensemble. Optogenetic suppression of prefrontal inputs to V1 reduced the contextual selectivity of deviance-detecting ensembles, demonstrating a causal role for top-down inputs. The presence of specialized context-selective ensembles in primary sensory cortex, modulated by higher cortical areas, provides a circuit substrate for the brain's construction and selection of prediction errors, computations which are key for survival and deficient in many psychiatric disorders.

Prolonged anesthesia alters brain synaptic architecture.

Prolonged medically induced coma (pMIC) is carried out routinely in intensive care medicine. pMIC leads to cognitive impairment, yet the underlying neuromorphological correlates are still unknown, as no direct studies of MIC exceeding ∼6 h on neural circuits exist. Here, we establish pMIC (up to 24 h) in adolescent and mature mice, and combine longitudinal two-photon imaging of cortical synapses with repeated behavioral object recognition assessments. We find that pMIC affects object recognition, and that it is associated with enhanced synaptic turnover, generated by enhanced synapse formation during pMIC, while the postanesthetic period is dominated by synaptic loss. Our results demonstrate major side effects of prolonged anesthesia on neural circuit structure.

Aberration-free holographic microscope for simultaneous imaging and stimulation of neuronal populations.

A technical challenge in neuroscience is to record and specifically manipulate the activity of neurons in living animals. This can be achieved in some preparations with two-photon calcium imaging and photostimulation. These methods can be extended to three dimensions by holographic light sculpting with spatial light modulators (SLMs). At the same time, performing simultaneous holographic imaging and photostimulation is still cumbersome, requiring two light paths with separate SLMs. Here we present an integrated optical design using a single SLM for simultaneous imaging and photostimulation. Furthermore, we applied axially dependent adaptive optics to make the system aberration-free, and developed software for calibrations and closed-loop neuroscience experiments. Finally, we demonstrate the performance of the system with simultaneous calcium imaging and optogenetics in mouse primary auditory cortex in vivo. Our integrated holographic system could facilitate the systematic investigation of neural circuit function in awake behaving animals.

Super-multiplex vibrational imaging.

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a 'colour barrier', owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

Identification of Pattern Completion Neurons in Neuronal Ensembles Using Probabilistic Graphical Models.

Neuronal ensembles are groups of neurons with coordinated activity that could represent sensory, motor, or cognitive states. The study of how neuronal ensembles are built, recalled, and involved in the guiding of complex behaviors has been limited by the lack of experimental and analytical tools to reliably identify and manipulate neurons that have the ability to activate entire ensembles. Such pattern completion neurons have also been proposed as key elements of artificial and biological neural networks. Indeed, the relevance of pattern completion neurons is highlighted by growing evidence that targeting them can activate neuronal ensembles and trigger behavior. As a method to reliably detect pattern completion neurons, we use conditional random fields (CRFs), a type of probabilistic graphical model. We apply CRFs to identify pattern completion neurons in ensembles in experiments using in vivo two-photon calcium imaging from primary visual cortex of male mice and confirm the CRFs predictions with two-photon optogenetics. To test the broader applicability of CRFs we also analyze publicly available calcium imaging data (Allen Institute Brain Observatory dataset) and demonstrate that CRFs can reliably identify neurons that predict specific features of visual stimuli. Finally, to explore the scalability of CRFs we apply them to in silico network simulations and show that CRFs-identified pattern completion neurons have increased functional connectivity. These results demonstrate the potential of CRFs to characterize and selectively manipulate neural circuits.SIGNIFICANCE STATEMENT We describe a graph theory method to identify and optically manipulate neurons with pattern completion capability in mouse cortical circuits. Using calcium imaging and two-photon optogenetics in vivo we confirm that key neurons identified by this method can recall entire neuronal ensembles. This method could be broadly applied to manipulate neuronal ensemble activity to trigger behavior or for therapeutic applications in brain prostheses.

Tracking calcium dynamics from individual neurons in behaving animals.

Measuring the activity of neuronal populations with calcium imaging can capture emergent functional properties of neuronal circuits with single cell resolution. However, the motion of freely behaving animals, together with the intermittent detectability of calcium sensors, can hinder automatic monitoring of neuronal activity and their subsequent functional characterization. We report the development and open-source implementation of a multi-step cellular tracking algorithm (Elastic Motion Correction and Concatenation or EMC2) that compensates for the intermittent disappearance of moving neurons by integrating local deformation information from detectable neurons. We demonstrate the accuracy and versatility of our algorithm using calcium imaging data from two-photon volumetric microscopy in visual cortex of awake mice, and from confocal microscopy in behaving Hydra, which experiences major body deformation during its contractions. We quantify the performance of our algorithm using ground truth manual tracking of neurons, along with synthetic time-lapse sequences, covering a wide range of particle motions and detectability parameters. As a demonstration of the utility of the algorithm, we monitor for several days calcium activity of the same neurons in layer 2/3 of mouse visual cortex in vivo, finding significant turnover within the active neurons across days, with only few neurons that remained active across days. Also, combining automatic tracking of single neuron activity with statistical clustering, we characterize and map neuronal ensembles in behaving Hydra, finding three major non-overlapping ensembles of neurons (CB, RP1 and RP2) whose activity correlates with contractions and elongations. Our results show that the EMC2 algorithm can be used as a robust and versatile platform for neuronal tracking in behaving animals.

Electrical compartmentalization in dendritic spines.

Most excitatory inputs in the CNS contact dendritic spines, avoiding dendritic shafts, so spines must play a key role for neurons. Recent data suggest that, in addition to enhancing connectivity and isolating synaptic biochemistry, spines can behave as electrical compartments independent from their parent dendrites. It is becoming clear that, although spines experience voltages similar to those of dendrites during action potentials (APs), spines must sustain higher depolarizations than do dendritic shafts during excitatory postsynaptic potentials (EPSPs). Synaptic potentials are likely amplified at the spine head and then reduced as they invade the dendrite through the spine neck. These electrical changes, probably due to a combination of passive and active mechanisms, may prevent the saturation of dendrites by the joint activation of many inputs, influence dendritic integration, and contribute to rapid synaptic plasticity. The electrical properties of spines could enable neural circuits to harness a high connectivity, implementing a "synaptic democracy," where each input can be individually integrated, tallied, and modified in order to generate emergent functional states.

Holographic Imaging and Stimulation of Neural Circuits.

A critical neuroscience challenge is the need to optically image and manipulate neural activity with high spatiotemporal resolution over large brain volumes. The last three decades have seen the development of calcium imaging to record activity from neuronal populations, as well as optochemistry and optogenetics to optically manipulate neural activity. These methods are typically implemented with wide-field or laser-scanning microscopes. While the former approach has a good temporal resolution, it generally lacks spatial resolution or specificity, particularly in scattering tissues such as the nervous system; meanwhile, the latter approach, particularly when combined with two-photon excitation, has high spatial resolution and specificity but poor temporal resolution. As a new technique, holographic microscopy combines the advantages of both approaches. By projecting a holographic pattern on the brain through a spatial light modulator, the activity of specific groups of neurons in 3D brain volumes can be imaged or stimulated with high spatiotemporal resolution. In a combination of other techniques such as fast scanning or temporal focusing, this high spatiotemporal resolution can be further improved. Holographic microscopy enables all-optical interrogating of neural activity in 3D, a critical tool to dissect the function of neural circuits.

Acute Focal Seizures Start As Local Synchronizations of Neuronal Ensembles.

Understanding seizure formation and spread remains a critical goal of epilepsy research. We used fast in vivo two-photon calcium imaging in male mouse neocortex to reconstruct, with single-cell resolution, the dynamics of acute (4-aminopyridine) focal cortical seizures as they originate within a spatially confined seizure initiation site (intrafocal region), and subsequently propagate into neighboring cortical areas (extrafocal region). We find that seizures originate as local neuronal ensembles within the initiation site. This abnormal hyperactivity engages increasingly larger areas in a saltatory fashion until it breaks into neighboring cortex, where it proceeds smoothly and is then detected electrophysiologically (LFP). Interestingly, PV inhibitory interneurons have spatially heterogeneous activity in intrafocal and extrafocal territories, ruling out a simple role of inhibition in seizure formation and spread. We propose a two-step model for the progression of focal seizures, where neuronal ensembles activate first, generating a microseizure, followed by widespread neural activation in a traveling wave through neighboring cortex during macroseizures.SIGNIFICANCE STATEMENT We have used calcium imaging in mouse sensory cortex in vivo to reconstruct the onset of focal seizures elicited by local injection of the chemoconvulsant 4-aminopyridine. We demonstrate at cellular resolution that acute focal seizures originate as increasingly synchronized local neuronal ensembles. Because of its spatial confinement, this process may at first be undetectable even by nearby LFP electrodes. Further, we establish spatial footprints of local neural subtype activity that correspond to consecutive steps of seizure microprogression. Such footprints could facilitate determining the recording location (e.g., inside/outside an epileptogenic focus) in high-resolution studies, even in the absence of a priori knowledge about where exactly a seizure started.

In vivo imaging of neural activity.

Since the introduction of calcium imaging to monitor neuronal activity with single-cell resolution, optical imaging methods have revolutionized neuroscience by enabling systematic recordings of neuronal circuits in living animals. The plethora of methods for functional neural imaging can be daunting to the nonexpert to navigate. Here we review advanced microscopy techniques for in vivo functional imaging and offer guidelines for which technologies are best suited for particular applications.

From the neuron doctrine 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.