Survey of spiking in the mouse visual system reveals functional hierarchy.

The anatomy of the mammalian visual system, from the retina to the neocortex, is organized hierarchically1. However, direct observation of cellular-level functional interactions across this hierarchy is lacking due to the challenge of simultaneously recording activity across numerous regions. Here we describe a large, open dataset-part of the Allen Brain Observatory2-that surveys spiking from tens of thousands of units in six cortical and two thalamic regions in the brains of mice responding to a battery of visual stimuli. Using cross-correlation analysis, we reveal that the organization of inter-area functional connectivity during visual stimulation mirrors the anatomical hierarchy from the Allen Mouse Brain Connectivity Atlas3. We find that four classical hierarchical measures-response latency, receptive-field size, phase-locking to drifting gratings and response decay timescale-are all correlated with the hierarchy. Moreover, recordings obtained during a visual task reveal that the correlation between neural activity and behavioural choice also increases along the hierarchy. Our study provides a foundation for understanding coding and signal propagation across hierarchically organized cortical and thalamic visual areas.

Removing independent noise in systems neuroscience data using DeepInterpolation.

Progress in many scientific disciplines is hindered by the presence of independent noise. Technologies for measuring neural activity (calcium imaging, extracellular electrophysiology and functional magnetic resonance imaging (fMRI)) operate in domains in which independent noise (shot noise and/or thermal noise) can overwhelm physiological signals. Here, we introduce DeepInterpolation, a general-purpose denoising algorithm that trains a spatiotemporal nonlinear interpolation model using only raw noisy samples. Applying DeepInterpolation to two-photon calcium imaging data yielded up to six times more neuronal segments than those computed from raw data with a 15-fold increase in the single-pixel signal-to-noise ratio (SNR), uncovering single-trial network dynamics that were previously obscured by noise. Extracellular electrophysiology recordings processed with DeepInterpolation yielded 25% more high-quality spiking units than those computed from raw data, while DeepInterpolation produced a 1.6-fold increase in the SNR of individual voxels in fMRI datasets. Denoising was attained without sacrificing spatial or temporal resolution and without access to ground truth training data. We anticipate that DeepInterpolation will provide similar benefits in other domains in which independent noise contaminates spatiotemporally structured datasets.

Columnar Localization and Laminar Origin of Cortical Surface Electrical Potentials.

Electrocorticography (ECoG) methodologically bridges basic neuroscience and understanding of human brains in health and disease. However, the localization of ECoG signals across the surface of the brain and the spatial distribution of their generating neuronal sources are poorly understood. To address this gap, we recorded from rat auditory cortex using customized µECoG, and simulated cortical surface electrical potentials with a full-scale, biophysically detailed cortical column model. Experimentally, µECoG-derived auditory representations were tonotopically organized and signals were anisotropically localized to less than or equal to ±200 µm, that is, a single cortical column. Biophysical simulations reproduce experimental findings and indicate that neurons in cortical layers V and VI contribute ∼85% of evoked high-gamma signal recorded at the surface. Cell number and synchrony were the primary biophysical properties determining laminar contributions to evoked µECoG signals, whereas distance was only a minimal factor. Thus, evoked µECoG signals primarily originate from neurons in the infragranular layers of a single cortical column.SIGNIFICANCE STATEMENT ECoG methodologically bridges basic neuroscience and understanding of human brains in health and disease. However, the localization of ECoG signals across the surface of the brain and the spatial distribution of their generating neuronal sources are poorly understood. We investigated the localization and origins of sensory-evoked ECoG responses. We experimentally found that ECoG responses were anisotropically localized to a cortical column. Biophysically detailed simulations revealed that neurons in layers V and VI were the primary sources of evoked ECoG responses. These results indicate that evoked ECoG high-gamma responses are primarily generated by the population spike rate of pyramidal neurons in layers V and VI of single cortical columns and highlight the possibility of understanding how microscopic sources produce mesoscale signals.

Fully integrated silicon probes for high-density recording of neural activity.

Sensory, motor and cognitive operations involve the coordinated action of large neuronal populations across multiple brain regions in both superficial and deep structures. Existing extracellular probes record neural activity with excellent spatial and temporal (sub-millisecond) resolution, but from only a few dozen neurons per shank. Optical Ca2+ imaging offers more coverage but lacks the temporal resolution needed to distinguish individual spikes reliably and does not measure local field potentials. Until now, no technology compatible with use in unrestrained animals has combined high spatiotemporal resolution with large volume coverage. Here we design, fabricate and test a new silicon probe known as Neuropixels to meet this need. Each probe has 384 recording channels that can programmably address 960 complementary metal-oxide-semiconductor (CMOS) processing-compatible low-impedance TiN sites that tile a single 10-mm long, 70 × 20-µm cross-section shank. The 6 × 9-mm probe base is fabricated with the shank on a single chip. Voltage signals are filtered, amplified, multiplexed and digitized on the base, allowing the direct transmission of noise-free digital data from the probe. The combination of dense recording sites and high channel count yielded well-isolated spiking activity from hundreds of neurons per probe implanted in mice and rats. Using two probes, more than 700 well-isolated single neurons were recorded simultaneously from five brain structures in an awake mouse. The fully integrated functionality and small size of Neuropixels probes allowed large populations of neurons from several brain structures to be recorded in freely moving animals. This combination of high-performance electrode technology and scalable chip fabrication methods opens a path towards recording of brain-wide neural activity during behaviour.

Two-photon frequency division multiplexing for functional in vivo imaging: a feasibility study.

Recently, we presented a new approach to create high-speed amplitude modulation of femtosecond laser pulses and tag multiple excitation beams with specific modulation frequencies. In this work, we discuss the utility of this method to record calcium signals in brain tissue with two-photon frequency-division multiplexing (2P-FDM) microscopy. While frequency-multiplexed imaging appears slightly inferior in terms of image quality as compared to conventional two-photon laser scanning microscopy due to shot noise-induced cross-talk between frequency channels, applying this technique to record average signals from regions of interest (ROI) such as neuronal cell bodies was found to be promising. We use phase information associated with each pixel or waveform within a selected ROI to phase-align and recombine the signals into one extended amplitude-modulated waveform. This procedure narrows the frequency detection window, effectively decreasing noise contributions from other frequency channels. Using theoretical analysis, numerical simulations, and in vitro imaging, we demonstrate a reduction of cross-talk by more than an order of magnitude and predict the usefulness of 2P-FDM for functional studies of brain activity.

Saccade-Responsive Visual Cortical Neurons Do Not Exhibit Distinct Visual Response Properties.

Rapid saccadic eye movements are used by animals to sample different parts of the visual scene. Previous work has investigated neural correlates of these saccades in visual cortical areas such as V1; however, how saccade-responsive neurons are distributed across visual areas, cell types, and cortical layers has remained unknown. Through analyzing 818 1 h experimental sessions from the Allen Brain Observatory, we present a large-scale analysis of saccadic behaviors in head-fixed mice and their neural correlates. We find that saccade-responsive neurons are present across visual cortex, but their distribution varies considerably by transgenically defined cell type, cortical area, and cortical layer. We also find that saccade-responsive neurons do not exhibit distinct visual response properties from the broader neural population, suggesting that the saccadic responses of these neurons are likely not predominantly visually driven. These results provide insight into the roles played by different cell types within a broader, distributed network of sensory and motor interactions.

Relationship between simultaneously recorded spiking activity and fluorescence signal in GCaMP6 transgenic mice.

Fluorescent calcium indicators are often used to investigate neural dynamics, but the relationship between fluorescence and action potentials (APs) remains unclear. Most APs can be detected when the soma almost fills the microscope's field of view, but calcium indicators are used to image populations of neurons, necessitating a large field of view, generating fewer photons per neuron, and compromising AP detection. Here, we characterized the AP-fluorescence transfer function in vivo for 48 layer 2/3 pyramidal neurons in primary visual cortex, with simultaneous calcium imaging and cell-attached recordings from transgenic mice expressing GCaMP6s or GCaMP6f. While most APs were detected under optimal conditions, under conditions typical of population imaging studies, only a minority of 1 AP and 2 AP events were detected (often <10% and ~20-30%, respectively), emphasizing the limits of AP detection under more realistic imaging conditions.

Neurons, the cells that make up the nervous system, transmit information using electrical signals known as action potentials or spikes. Studying the spiking patterns of neurons in the brain is essential to understand perception, memory, thought, and behaviour. One way to do that is by recording electrical activity with microelectrodes. Another way to study neuronal activity is by using molecules that change how they interact with light when calcium binds to them, since changes in calcium concentration can be indicative of neuronal spiking. That change can be observed with specialized microscopes know as two-photon fluorescence microscopes. Using calcium indicators, it is possible to simultaneously record hundreds or even thousands of neurons. However, calcium fluorescence and spikes do not translate one-to-one. In order to interpret fluorescence data, it is important to understand the relationship between the fluorescence signals and the spikes associated with individual neurons. The only way to directly measure this relationship is by using calcium imaging and electrical recording simultaneously to record activity from the same neuron. However, this is extremely challenging experimentally, so this type of data is rare. To shed some light on this, Huang, Ledochowitsch et al. used mice that had been genetically modified to produce a calcium indicator in neurons of the visual cortex and simultaneously obtained both fluorescence measurements and electrical recordings from these neurons. These experiments revealed that, while the majority of time periods containing multi-spike neural activity could be identified using calcium imaging microscopy, on average, less than 10% of isolated single spikes were detectable. This is an important caveat that researchers need to take into consideration when interpreting calcium imaging results. These findings are intended to serve as a guide for interpreting calcium imaging studies that look at neurons in the mammalian brain at the population level. In addition, the data provided will be useful as a reference for the development of activity sensors, and to benchmark and improve computational approaches for detecting and predicting spikes.

Reconciling functional differences in populations of neurons recorded with two-photon imaging and electrophysiology.

Extracellular electrophysiology and two-photon calcium imaging are widely used methods for measuring physiological activity with single-cell resolution across large populations of cortical neurons. While each of these two modalities has distinct advantages and disadvantages, neither provides complete, unbiased information about the underlying neural population. Here, we compare evoked responses in visual cortex recorded in awake mice under highly standardized conditions using either imaging of genetically expressed GCaMP6f or electrophysiology with silicon probes. Across all stimulus conditions tested, we observe a larger fraction of responsive neurons in electrophysiology and higher stimulus selectivity in calcium imaging, which was partially reconciled by applying a spikes-to-calcium forward model to the electrophysiology data. However, the forward model could only reconcile differences in responsiveness when restricted to neurons with low contamination and an event rate above a minimum threshold. This work established how the biases of these two modalities impact functional metrics that are fundamental for characterizing sensory-evoked responses.

Experience shapes activity dynamics and stimulus coding of VIP inhibitory cells.

Cortical circuits can flexibly change with experience and learning, but the effects on specific cell types, including distinct inhibitory types, are not well understood. Here we investigated how excitatory and VIP inhibitory cells in layer 2/3 of mouse visual cortex were impacted by visual experience in the context of a behavioral task. Mice learned a visual change detection task with a set of eight natural scene images. Subsequently, during 2-photon imaging experiments, mice performed the task with these familiar images and three sets of novel images. Strikingly, the temporal dynamics of VIP activity differed markedly between novel and familiar images: VIP cells were stimulus-driven by novel images but were suppressed by familiar stimuli and showed ramping activity when expected stimuli were omitted from a temporally predictable sequence. This prominent change in VIP activity suggests that these cells may adopt different modes of processing under novel versus familiar conditions.

A large-scale standardized physiological survey reveals functional organization of the mouse visual cortex.

To understand how the brain processes sensory information to guide behavior, we must know how stimulus representations are transformed throughout the visual cortex. Here we report an open, large-scale physiological survey of activity in the awake mouse visual cortex: the Allen Brain Observatory Visual Coding dataset. This publicly available dataset includes the cortical activity of nearly 60,000 neurons from six visual areas, four layers, and 12 transgenic mouse lines in a total of 243 adult mice, in response to a systematic set of visual stimuli. We classify neurons on the basis of joint reliabilities to multiple stimuli and validate this functional classification with models of visual responses. While most classes are characterized by responses to specific subsets of the stimuli, the largest class is not reliably responsive to any of the stimuli and becomes progressively larger in higher visual areas. These classes reveal a functional organization wherein putative dorsal areas show specialization for visual motion signals.

A Large-Scale Interface for Optogenetic Stimulation and Recording in Nonhuman Primates.

While optogenetics offers great potential for linking brain function and behavior in nonhuman primates, taking full advantage of that potential will require stable access for optical stimulation and concurrent monitoring of neural activity. Here we present a practical, stable interface for stimulation and recording of large-scale cortical circuits. To obtain optogenetic expression across a broad region, here spanning primary somatosensory (S1) and motor (M1) cortices, we used convection-enhanced delivery of the viral vector, with online guidance from MRI. To record neural activity across this region, we used a custom micro-electrocorticographic (µECoG) array designed to minimally attenuate optical stimuli. Lastly, we demonstrated the use of this interface to measure spatiotemporal responses to optical stimulation across M1 and S1. This interface offers a powerful tool for studying circuit dynamics and connectivity across cortical areas, for long-term studies of neuromodulation and targeted cortical plasticity, and for linking these to behavior.

A transparent µECoG array for simultaneous recording and optogenetic stimulation.

In this paper we report for the first time the design, fabrication and characterization of an optically transparent electrode array for micro-electrocorticography. We present a 49-channel µECoG array with an electrode pitch of 800 µm and a 16-channel linear µECoG array with an electrode pitch of 200 µm. The backing material was Parylene C. Transparent, sputtered indium tin oxide (ITO) was used in conjunction with e-beam evaporated gold to fabricate the electrodes. We provide electrochemical impedance characterization and light transmission data for the fabricated devices.