Joshua H. Siegle’s research while affiliated with Allen Institute for Brain Science and other places

Significance Compact tools capable of delivering multicolor optogenetic stimulation to deep tissue targets with sufficient span, spatiotemporal resolution, and optical power remain challenging to realize. Here, we demonstrate foundry-fabricated nanophotonic neural probes for blue and red photostimulation and electrophysiological recording, which use a combination of spatial multiplexing and on-shank wavelength demultiplexing to increase the number of on-shank emitters. Aim We demonstrate silicon (Si) photonic neural probes with 26 photonic channels and 26 recording sites, which were fabricated on 200-mm diameter wafers at a commercial Si photonics foundry. Each photonic channel consists of an on-shank demultiplexer and separate grating coupler emitters for blue and red light, for a total of 52 emitters. Approach We evaluate neural probe functionality through bench measurements and in vivo experiments by photostimulating through 16 of the available 26 emitter pairs. Results We report neural probe electrode impedances, optical transmission, and beam profiles. We validated a packaged neural probe in optogenetic experiments with mice sensitive to blue or red photostimulation. Conclusions Our foundry-fabricated nanophotonic neural probe demonstrates dense dual-color emitter integration on a single shank for targeted photostimulation. Given its two emission wavelengths, high emitter density, and long site span, this probe will facilitate experiments involving bidirectional circuit manipulations across both shallow and deep structures simultaneously.

The onset of a sensory stimulus elicits transient bursts of activity in neural populations, which are presumed to convey information about the stimulus to downstream populations. The time at which these synchronized bursts reach their peak is highly variable across stimulus presentations, but the relative timing of bursts across interconnected brain regions may be less variable, especially for regions that are strongly functionally coupled. We developed a simple analytical framework that obtains good estimates of population burst times on a trial-by-trial basis, and of the correlations in the timing of evoked population bursts across areas. We show that this method performs well on simulated data, and is 85 to 90% faster than an alternative, recently-published method while also being much easier to apply. Using this new approach, we examined the relative timing of the first two population bursts following the onset of a drifting grating stimulus in large-scale recordings of spiking activity from six cortical visual areas and one visual thalamic nucleus in thirteen mice. The new method allowed us to identify mouse-to-mouse variation in peak times and region-to-region functional coupling. While all results were consistent with known anatomy and physiology, we found some sequences of activity across areas to be the same across all mice, while others varied with the individual. The general approach can thus produce sensitive analyses of timing relationships across neural populations. Significant Statement Careful analysis can reveal strong and precisely-timed interactions across multiple brain areas from small populations of spiking neurons. We developed a computationally efficient procedure that allowed us to examine the relative timing and coupling of 7 visual areas (6 cortical and one thalamic) and compare results in over 10 mice. The method can be used to track the flow of information across the brain in response to stimuli or during a behavioral task.

Influenced by non-stationary factors such as brain states and behavior, neurons exhibit substantial response variability even to identical stimuli. However, it remains unclear how their relative impact on neuronal variability evolves over time. To address this question, we designed an encoding model conditioned on latent states to partition variability in the mouse visual cortex across internal brain dynamics, behavior, and external visual stimulus. Applying a hidden Markov model to local field potentials, we consistently identified three distinct oscillation states, each with a unique variability profile. Regression models within each state revealed a dynamic composition of factors influencing spiking variability, with the dominant factor switching within seconds. The state-conditioned regression model uncovered extensive diversity in source contributions across units, varying in accordance with anatomical hierarchy and internal state. This heterogeneity in encoding underscores the importance of partitioning variability over time, particularly when considering the influence of non-stationary factors on sensory processing.

High-resolution extracellular electrophysiology is the gold standard for recording spikes from distributed neural populations, and is especially powerful when combined with optogenetics for manipulation of specific cell types with high temporal resolution. We integrated these approaches into prototype Neuropixels Opto probes, which combine electronic and photonic circuits. These devices pack 960 electrical recording sites and two sets of 14 light emitters onto a 1 cm shank, allowing spatially addressable optogenetic stimulation with blue and red light. In mouse cortex, Neuropixels Opto probes delivered high-quality recordings together with spatially addressable optogenetics, differentially activating or silencing neurons at distinct cortical depths. In mouse striatum and other deep structures, Neuropixels Opto probes delivered efficient optotagging, facilitating the identification of two cell types in parallel. Neuropixels Opto probes represent an unprecedented tool for recording, identifying, and manipulating neuronal populations.

Behavioral neuroscience faces two conflicting demands: long-duration recordings from large neural populations and unimpeded animal behavior. To meet this challenge we developed ONIX, an open-source data acquisition system with high data throughput (2 GB s⁻¹) and low closed-loop latencies (<1 ms) that uses a 0.3-mm thin tether to minimize behavioral impact. Head position and rotation are tracked in three dimensions and used to drive active commutation without torque measurements. ONIX can acquire data from combinations of passive electrodes, Neuropixels probes, head-mounted microscopes, cameras, three-dimensional trackers and other data sources. We performed uninterrupted, long (~7 h) neural recordings in mice as they traversed complex three-dimensional terrain, and multiday sleep-tracking recordings (~55 h). ONIX enabled exploration with similar mobility as nonimplanted animals, in contrast to conventional tethered systems, which have restricted movement. By combining long recordings with full mobility, our technology will enable progress on questions that require high-quality neural recordings during ethologically grounded behaviors.

Significance: Compact tools capable of delivering multicolor optogenetic stimulation to deep tissue targets with sufficient span, spatiotemporal resolution, and optical power remain challenging to realize. Here, we demonstrate foundry-fabricated nanophotonic neural probes for blue and red photostimulation and electrophysiological recording, which use a combination of spatial multiplexing and on-shank wavelength-demultiplexing to increase the number of on-shank emitters. Aim: We demonstrate Si photonic neural probes with 26 photonic channels and 26 recording sites, which were fabricated on 200-mm diameter wafers at a commercial Si photonics foundry. Each photonic channel consists of an on-shank demultiplexer and separate grating coupler emitters for blue and red light, for a total of 52 emitters. Approach: We evaluate neural probe functionality through bench measurements and in vivo experiments by photostimulating through 16 of the available 26 emitter pairs. Results: We report neural probe electrode impedances, optical transmission, and beam profiles. We validated a packaged neural probe in optogenetic experiments with mice sensitive to blue or red photostimulation. Conclusions: Our foundry-fabricated nanophotonic neural probe demonstrates dense dual-color emitter integration on a single shank for targeted photostimulation. Given its two emission wavelengths, high emitter density, and long site span, this probe will facilitate experiments involving bidirectional circuit manipulations across both shallow and deep structures simultaneously.

Influenced by factors such as brain states and behavior, neurons exhibit substantial response variability even to identical stimuli. Because these factors are non-stationary, they dynamically impact the fidelity of sensory processing. However, it remains unclear how their relative impact on neuronal variability evolves over time. To address this question, we designed an encoding model with latent states to partition visual cortical variability across three crucial categories of sources: internal brain dynamics, behavior, and external visual stimulus. Applying a hidden Markov model to the rhythmic patterns of cortical local field potentials, we consistently identified three distinct oscillation states. Each state revealed a unique variability profile and a consistent descending trend of stimulus modulation across the visual hierarchy. Regression models within each state revealed a dynamic composition of factors contributing to the observed spiking variability, with the primary influencing factor switching within seconds. In the state dominated by high-frequency oscillations, sensory inputs and behavior exerted the most influence on population dynamics. Conversely, internal brain activity explained most of the variance in the state dominated by low-frequency oscillations. This heterogeneity across states underscores the importance of partitioning variability over time, particularly when considering the dynamic influence of non-stationary factors on sensory processing.

Targeting deep brain structures during electrophysiology and injections requires intensive training and expertise. Even with experience, researchers often can’t be certain that a probe is placed precisely in a target location and this complexity scales with the number of simultaneous probes used in an experiment. Here, we present Pinpoint, open-source software that allows for interactive exploration of stereotaxic insertion plans. Once an insertion plan is created, Pinpoint allows users to save these online and share them with collaborators. 3D modeling tools allow users to explore their insertions alongside rig and implant hardware and ensure plans are physically possible. Probes in Pinpoint can be linked to electronic micro-manipulators allowing real-time visualization of current brain region targets alongside neural data. In addition, Pinpoint can control manipulators to automate and parallelize the insertion process. Compared to previously available software, Pinpoint’s easy access through web browsers, extensive features, and real-time experiment integration enable more efficient and reproducible recordings.