Prospective Cohort Study

Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking. NpHR is compatible with ChR2, the previous optical excitation technology we have described, in that the two opposing probes operate at similar light powers but with well-separated action spectra. NpHR, like ChR2, functions in mammals without exogenous cofactors, and the two probes can be integrated with calcium imaging in mammalian brain tissue for bidirectional optical modulation and readout of neural activity. Likewise, NpHR and ChR2 can be targeted together to Caenorhabditis elegans muscle and cholinergic motor neurons to control locomotion bidirectionally. NpHR and ChR2 form a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.

Multimodal fast optical interrogation of neural circuitry

Neuronal populations in sensory cortex produce variable responses to sensory stimuli and exhibit intricate spontaneous activity even without external sensory input. Cortical variability and spontaneous activity have been variously proposed to represent random noise, recall of prior experience, or encoding of ongoing behavioral and cognitive variables. Recording more than 10,000 neurons in mouse visual cortex, we observed that spontaneous activity reliably encoded a high-dimensional latent state, which was partially related to the mouse's ongoing behavior and was represented not just in visual cortex but also across the forebrain. Sensory inputs did not interrupt this ongoing signal but added onto it a representation of external stimuli in orthogonal dimensions. Thus, visual cortical population activity, despite its apparently noisy structure, reliably encodes an orthogonal fusion of sensory and multidimensional behavioral information.

/pdf/spontaneous-behaviors-drive-multidimensional-brainwide-2vagih8taw.pdf

Spontaneous behaviors drive multidimensional, brainwide activity.

When experts are immersed in a task, do their brains prioritize task-related activity? Most efforts to understand neural activity during well-learned tasks focus on cognitive computations and task-related movements. We wondered whether task-performing animals explore a broader movement landscape and how this impacts neural activity. We characterized movements using video and other sensors and measured neural activity using widefield and two-photon imaging. Cortex-wide activity was dominated by movements, especially uninstructed movements not required for the task. Some uninstructed movements were aligned to trial events. Accounting for them revealed that neurons with similar trial-averaged activity often reflected utterly different combinations of cognitive and movement variables. Other movements occurred idiosyncratically, accounting for trial-by-trial fluctuations that are often considered 'noise'. This held true throughout task-learning and for extracellular Neuropixels recordings that included subcortical areas. Our observations argue that animals execute expert decisions while performing richly varied, uninstructed movements that profoundly shape neural activity.

/pdf/single-trial-neural-dynamics-are-dominated-by-richly-varied-19nzuvd349.pdf

Single-trial neural dynamics are dominated by richly varied movements

The human brain contains billions of cells called neurons that rapidly carry information from one part of the brain to another. Progress in medical research and healthcare is hindered by the difficulty in understanding precisely which neurons are active at any given time. New brain imaging techniques and genetic tools allow researchers to track the activity of thousands of neurons in living animals over many months. However, these experiments produce large volumes of data that researchers currently have to analyze manually, which can take a long time and generate irreproducible results. There is a need to develop new computational tools to analyze such data. The new tools should be able to operate on standard computers rather than just specialist equipment as this would limit the use of the solutions to particularly well-funded research teams. Ideally, the tools should also be able to operate in real-time as several experimental and therapeutic scenarios, like the control of robotic limbs, require this. To address this need, Giovannucci et al. developed a new software package called CaImAn to analyze brain images on a large scale. Firstly, the team developed algorithms that are suitable to analyze large sets of data on laptops and other standard computing equipment. These algorithms were then adapted to operate online in real-time. To test how well the new software performs against manual analysis by human researchers, Giovannucci et al. asked several trained human annotators to identify active neurons that were round or donut-shaped in several sets of imaging data from mouse brains. Each set of data was independently analyzed by three or four researchers who then discussed any neurons they disagreed on to generate a ‘consensus annotation’. Giovannucci et al. then used CaImAn to analyze the same sets of data and compared the results to the consensus annotations. This demonstrated that CaImAn is nearly as good as human researchers at identifying active neurons in brain images. CaImAn provides a quicker method to analyze large sets of brain imaging data and is currently used by over a hundred laboratories across the world. The software is open source, meaning that it is freely-available and that users are encouraged to customize it and collaborate with other users to develop it further.

CaImAn an open source tool for scalable calcium imaging data analysis

Two-photon microscopy of calcium-dependent sensors has enabled unprecedented recordings from vast populations of neurons. While the sensors and microscopes have matured over several generations of development, computational methods to process the resulting movies remain inefficient and can give results that are hard to interpret. Here we introduce Suite2p: a fast, accurate and complete pipeline that registers raw movies, detects active cells, extracts their calcium traces and infers their spike times. Suite2p runs on standard workstations, operates faster than real time, and recovers ~2 times more cells than the previous state-of-the-art method. Its low computational load allows routine detection of ~10,000 cells simultaneously with standard two-photon resonant-scanning microscopes. Recordings at this scale promise to reveal the fine structure of activity in large populations of neurons or large populations of subcellular structures such as synaptic boutons.

/pdf/suite2p-beyond-10-000-neurons-with-standard-two-photon-3042enp0y5.pdf

Suite2p: beyond 10,000 neurons with standard two-photon microscopy

Focal epilepsy involves excessive cortical activity that propagates both locally and distally. Does this propagation follow the same routes as normal cortical activity? We pharmacologically induced focal seizures in primary visual cortex (V1) of awake mice, and compared their propagation to the retinotopic organization of V1 and higher visual areas. We used simultaneous local field potential recordings and widefield imaging of a genetically encoded calcium indicator to measure prolonged seizures (ictal events) and brief interictal events. Both types of event are orders of magnitude larger than normal visual responses, and both start as standing waves: synchronous elevated activity in the V1 focus and in homotopic locations in higher areas, i.e. locations with matching retinotopic preference. Following this common beginning, however, seizures persist and propagate both locally and into homotopic distal regions, and eventually invade all of visual cortex and beyond. We conclude that seizure initiation resembles the initiation of interictal events, and seizure propagation respects the connectivity underlying normal visual processing. Focal cortical seizures result from local and widespread propagation of excitatory activity. Here the authors employ widefield calcium imaging in mouse visual areas to demonstrate that these seizures start as local synchronous activation and then propagate along the connectivity that underlies normal sensory processing.

/pdf/focal-cortical-seizures-start-as-standing-waves-and-179a5aqyz4.pdf

Focal cortical seizures start as standing waves and propagate respecting homotopic connectivity.

The selectivity of neuronal responses arises from the architecture of excitatory and inhibitory connections. In the primary visual cortex, the selectivity of a neuron in layer 2/3 for stimulus orientation and direction is thought to arise from intracortical inputs that are similarly selective1–8. However, the excitatory inputs of a neuron can have diverse stimulus preferences1–4,6,7,9, and inhibitory inputs can be promiscuous10 and unselective11. Here we show that the excitatory and inhibitory intracortical connections to a layer 2/3 neuron accord with its selectivity by obeying precise spatial patterns. We used rabies tracing1,12 to label and functionally image the excitatory and inhibitory inputs to individual pyramidal neurons of layer 2/3 of the mouse visual cortex. Presynaptic excitatory neurons spanned layers 2/3 and 4 and were distributed coaxial to the preferred orientation of the postsynaptic neuron, favouring the region opposite to its preferred direction. By contrast, presynaptic inhibitory neurons resided within layer 2/3 and favoured locations near the postsynaptic neuron and ahead of its preferred direction. The direction selectivity of a postsynaptic neuron was unrelated to the selectivity of presynaptic neurons, but correlated with the spatial displacement between excitatory and inhibitory presynaptic ensembles. Similar asymmetric connectivity establishes direction selectivity in the retina13–17. This suggests that this circuit motif might be canonical in sensory processing. In the mouse visual cortex, the excitatory and inhibitory presynaptic neurons of individual layer 2/3 pyramidal neurons are spatially offset to generate direction-selective responses.

Spatial connectivity matches direction selectivity in visual cortex.

Epilepsy research is rapidly adopting novel fluorescence optical imaging methods to tackle unresolved questions on the cellular and circuit mechanisms of seizure generation and evolution. State of the art two-photon microscopy and wide-field fluorescence imaging can record the activity in epileptic networks at multiple scales, from neuronal microcircuits to brain-wide networks. These approaches exploit transgenic and viral technologies to target genetically encoded calcium and voltage sensitive indicators to subclasses of neurons, and achieve genetic specificity, spatial resolution and scalability that can complement electrophysiological recordings from awake animal models of epilepsy. Two-photon microscopy is well suited to study single neuron dynamics during interictal and ictal events, and highlight the differences between the activity of excitatory and inhibitory neuronal classes in the focus and propagation zone. In contrast, wide-field fluorescence imaging provides mesoscopic recordings from the entire cortical surface, necessary to investigate seizure propagation pathways, and how the unfolding of epileptic events depends on the topology of brain-wide functional connectivity. Answering these questions will inform pre-clinical studies attempting to suppress seizures with gene therapy, optogenetic or chemogenetic strategies. Dissecting which network nodes outside the seizure onset zone are important for seizure generation, propagation and termination can be used to optimize current and future evaluation methods to identify an optimal surgical strategy.

/pdf/the-enlightened-brain-novel-imaging-methods-focus-on-3yh0o4mjbm.pdf

The Enlightened Brain: Novel Imaging Methods Focus on Epileptic Networks at Multiple Scales

Focal epilepsy involves excessive and synchronous cortical activity that propagates both locally and dis-tally. Does this propagation follow the same routes as normal cortical activity? We induced focal seizures in primary visual cortex (V1) of awake mice, and compared their propagation to the retinotopic organization of V1 and higher visual areas. We measured activity through simultaneous local field potential recordings and widefield imaging of a genetically encoded calcium indicator, and observed both prolonged seizures (ictal events) and brief interictal events. Both types of event were orders of magnitude larger than normal visual responses, and both started as standing waves: synchronous elevated activity in the focal V1 region and in corresponding retinotopic locations in higher areas. Following this common beginning, however, seizures, persisted and propagated both locally and into distal regions. These regions matched each other in retinotopy. We conclude that seizure propagation respects the functional connectivity underlying normal visual processing.

http://www.biorxiv.org/content/early/2016/10/12/080598.full.pdf

L. Federico Rossi

Papers

Suite2p: beyond 10,000 neurons with standard two-photon microscopy

Focal cortical seizures start as standing waves and propagate respecting homotopic connectivity.

Spatial connectivity matches direction selectivity in visual cortex.

The Enlightened Brain: Novel Imaging Methods Focus on Epileptic Networks at Multiple Scales

Cortical seizure propagation respects functional connectivity underlying sensory processing