Daniel J. Denman

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.

On Parallel Streams through the Mouse Dorsal Lateral Geniculate Nucleus

The mouse visual system is an emerging model for the study of cortical and thalamic circuit function. To maximize the usefulness of this model system, it is important to analyze the similarities and differences between the organization of all levels of the murid visual system with other, better studied systems (e.g., non-human primates and the domestic cat). While the understanding of mouse retina and cortex has expanded rapidly, less is known about mouse dorsal lateral geniculate nucleus (dLGN). Here, we study whether parallel processing streams exist in mouse dLGN. We use a battery of stimuli that have been previously shown to successfully distinguish parallel streams in other species: electrical stimulation of the optic chiasm, contrast-reversing stationary gratings at varying spatial phase, drifting sinusoidal gratings, dense noise for receptive field reconstruction, and frozen contrast-modulating noise. As in the optic nerves of domestic cats and non-human primates, we find evidence for multiple conduction velocity groups after optic chiasm stimulation. As in so-called “visual mammals”, we find a subpopulation of mouse dLGN cells showing non-linear spatial summation. However, differences in stimulus selectivity and sensitivity do not provide sufficient basis for identification of clearly distinct classes of relay cells. Nevertheless, consistent with presumptively homologous status of dLGNs of all mammals, there are substantial similarities between response properties of mouse dLGN neurons and those of cats and primates.

Spatial organization of chromatic pathways in the mouse dorsal lateral geniculate nucleus

In both dichromats and trichromats, cone opsin signals are maintained independently in cones and combined at the bipolar and retinal ganglion cell level, creating parallel color opponent pathways to the central visual system. Like other dichromats, the mouse retina expresses a short-wavelength (S) and a medium-wavelength (M) opsin, with the S-opsin shifted to peak sensitivity in the ultraviolet (UV) range. Unlike in primates, nonuniform opsin expression across the retina and coexpression in single cones creates a mostly mixed chromatic signal. Here, we describe the visuotopic and chromatic organization of spiking responses in the dorsal lateral geniculate and of the local field potentials in their recipient zone in primary visual cortex (V1). We used an immersive visual stimulus dome that allowed us to present spatiotemporally modulated UV and green luminance in any region of the visual field of an awake, head-fixed mouse. Consistent with retinal expression of opsins, we observed graded UV-to-green dominated responses from the upper to lower visual fields, with a smaller difference across azimuth. In addition, we identified a subpopulation of cells (<10%) that exhibited spectrally opponent responses along the S–M axis. Luminance signals of each wavelength and color signals project to the middle layers of V1. SIGNIFICANCE STATEMENT In natural environments, color information is useful for guiding behavior. How small terrestrial mammals such as mice use graded expression of cone opsins to extract visual information from their environments is not clear, even as the use of mice for studying visually guided behavior grows. In this study, we examined the color signals that the retina sends to the visual cortex via the lateral geniculate nucleus of the thalamus. We found that green dominated responses in the lower and nasal visual field and ultraviolet dominated responses in the upper visual field. We describe a subset of cells that exhibit color opponent responses.

Closed-loop, ultraprecise, automated craniotomies

A large array of neuroscientific techniques, including in vivo electrophysiology, two-photon imaging, optogenetics, lesions, and microdialysis, require access to the brain through the skull. Ideally, the necessary craniotomies could be performed in a repeatable and automated fashion, without damaging the underlying brain tissue. Here we report that when drilling through the skull a stereotypical increase in conductance can be observed when the drill bit passes through the skull base. We present an architecture for a robotic device that can perform this algorithm, along with two implementations--one based on homebuilt hardware and one based on commercially available hardware--that can automatically detect such changes and create large numbers of precise craniotomies, even in a single skull. We also show that this technique can be adapted to automatically drill cranial windows several millimeters in diameter. Such robots will not only be useful for helping neuroscientists perform both small and large craniotomies more reliably but can also be used to create precisely aligned arrays of craniotomies with stereotaxic registration to standard brain atlases that would be difficult to drill by hand.

From Maxwell's equations to the theory of current-source density analysis

Despite the widespread use of current‐source density (CSD) analysis of extracellular potential recordings in the brain, the physical mechanisms responsible for the generation of the signal are still debated. While the extracellular potential is thought to be exclusively generated by the transmembrane currents, recent studies suggest that extracellular diffusive, advective and displacement currents—traditionally neglected—may also contribute considerably toward extracellular potential recordings. Here, we first justify the application of the electro‐quasistatic approximation of Maxwells equations to describe the electromagnetic field of physiological origin. Subsequently, we perform spatial averaging of currents in neural tissue to arrive at the notion of the CSD and derive an equation relating it to the extracellular potential. We show that, in general, the extracellular potential is determined by the CSD of membrane currents as well as the gradients of the putative extracellular diffusion current. The diffusion current can contribute significantly to the extracellular potential at frequencies less than a few Hertz; in which case it must be subtracted to obtain correct CSD estimates. We also show that the advective and displacement currents in the extracellular space are negligible for physiological frequencies while, within cellular membrane, displacement current contributes toward the CSD as a capacitive current. Taken together, these findings elucidate the relationship between electric currents and the extracellular potential in brain tissue and form the necessary foundation for the analysis of extracellular recordings.

Ultracompact optoflex neural probes for high-resolution electrophysiology and optogenetic stimulation

We report on the development of high-density neural probes for distributed neuronal recording and stimulation. Our hybrid silicon-parylene probes provide high spatial resolution and incorporate a monolithically integrated flexible cable to address the challenge of stable recordings in chronic neural implants. We address a long-standing but often overlooked issue in parylene processing to realize reliable multilayer interconnects. We also discuss the design of ultracompact parylene optical waveguides for localized optogenetic stimulation of neurons. We demonstrate in-vivo electrophysiology recordings in mice.

Mouse color and wavelength-specific luminance contrast sensitivity are non-uniform across visual space

Mammalian visual behaviors, as well as responses in the neural systems underlying these behaviors, are driven by luminance and color contrast. With constantly improving tools for measuring activity in cell-type-specific populations in the mouse during visual behavior, it is important to define the extent of luminance and color information that is behaviorally accessible to the mouse. A non-uniform distribution of cone opsins in the mouse retina potentially complicates both luminance and color sensitivity; opposing gradients of short (UV-shifted) and middle (blue/green) cone opsins suggest that color discrimination and wavelength-specific luminance contrast sensitivity may differ with retinotopic location. Here we ask how well mice can discriminate color and wavelength-specific luminance changes across visuotopic space. We found that mice were able to discriminate color and were able to do so more broadly across visuotopic space than expected from the cone-opsin distribution. We also found wavelength-band-specific differences in luminance sensitivity.

Visual physiology of the Layer 4 cortical circuit in silico

Despite advances in experimental techniques and accumulation of large datasets concerning the composition and properties of the cortex, quantitative modeling of cortical circuits under in-vivo-like conditions remains challenging. Here we report and publicly release a biophysically detailed circuit model of layer 4 in the mouse primary visual cortex, receiving thalamo-cortical visual inputs. The 45,000-neuron model was subjected to a battery of visual stimuli, and results were compared to published work and new in vivo experiments. Simulations reproduced a variety of observations, including effects of optogenetic perturbations. Critical to the agreement between responses in silico and in vivo were the rules of functional synaptic connectivity between neurons. Interestingly, after extreme simplification the model still performed satisfactorily on many measurements, although quantitative agreement with experiments suffered. These results emphasize the importance of functional rules of cortical wiring and enable a next generation of data-driven models of in vivo neural activity and computations. AUTHOR SUMMARY How can we capture the incredible complexity of brain circuits in quantitative models, and what can such models teach us about mechanisms underlying brain activity? To answer these questions, we set out to build extensive, bio-realistic models of brain circuitry employing systematic datasets on brain structure and function. Here we report the first modeling results of this project, focusing on the layer 4 of the primary visual cortex (V1) of the mouse. Our simulations reproduced a variety of experimental observations in a large battery of visual stimuli. The results elucidated circuit mechanisms determining patters of neuronal activity in layer 4 – in particular, the roles of feedforward thalamic inputs and specific patterns of intracortical connectivity in producing tuning of neuronal responses to the orientation of motion. Simplification of neuronal models led to specific deficiencies in reproducing experimental data, giving insights into how biological details contribute to various aspects of brain activity. To enable future development of more sophisticated models, we make the software code, the model, and simulation results publicly available.

Optogenetics in Mice Performing a Visual Discrimination Task: Measurement and Suppression of Retinal Activation and the Resulting Behavioral Artifact

Optogenetic techniques are used widely to perturb and interrogate neural circuits in behaving animals, but illumination can have additional effects, such as the activation of endogenous opsins in the retina. We found that illumination, delivered deep into the brain via an optical fiber, evoked a behavioral artifact in mice performing a visually guided discrimination task. Compared with blue (473 nm) and yellow (589 nm) illumination, red (640 nm) illumination evoked a greater behavioral artifact and more activity in the retina, the latter measured with electrical recordings. In the mouse, the sensitivity of retinal opsins declines steeply with wavelength across the visible spectrum, but propagation of light through brain tissue increases with wavelength. Our results suggest that poor retinal sensitivity to red light was overcome by relatively robust propagation of red light through brain tissue and stronger illumination of the retina by red than by blue or yellow light. Light adaptation of the retina, via an external source of illumination, suppressed retinal activation and the behavioral artifact without otherwise impacting behavioral performance. In summary, long wavelength optogenetic stimuli are particularly prone to evoke behavioral artifacts via activation of retinal opsins in the mouse, but light adaptation of the retina can provide a simple and effective mitigation of the artifact.

High-density extracellular probes reveal dendritic backpropagation and facilitate neuron classification

Different neuron types serve distinct roles in neural processing. Extracellular electrical recordings are extensively used to study brain function but are typically blind to cell identity. Morpho-electric properties of neurons measured on spatially dense electrode arrays might be useful for distinguishing neuron types. Here we used Neuropixels probes to record from cortical and subcortical regions of the mouse brain. Extracellular waveforms of each neuron were detected across many channels and showed distinct spatiotemporal profiles among brain regions. Classification of neurons by brain region was improved with multi-channel compared to single-channel waveforms. In visual cortex, waveform clustering identified the canonical regular spiking (RS) and fast spiking (FS) classes, but also uncovered a subclass of RS units with unidirectional backpropagating action potentials (BAPs). Moreover, BAPs were observed in many hippocampal RS cells. Overall, waveform analysis of spikes from high-density probes aids neuron identification and can reveal dendritic backpropagation.