SHIELD: Skull-shaped hemispheric implants enabling large-scale electrophysiology datasets in the mouse brain.

To understand the neural basis of behavior, it is essential to measure spiking dynamics across many interacting brain regions. Although new technologies, such as Neuropixels probes, facilitate multi-regional recordings, significant surgical and procedural hurdles remain for these experiments to achieve their full potential. Here, we describe skull-shaped hemispheric implants enabling large-scale electrophysiology datasets (SHIELD). These 3D-printed skull-replacement implants feature customizable insertion holes, allowing dozens of cortical and subcortical structures to be recorded in a single mouse using repeated multi-probe insertions over many days. We demonstrate the procedure's high success rate, biocompatibility, lack of adverse effects on behavior, and compatibility with imaging and optogenetics. To showcase SHIELD's scientific utility, we use multi-probe recordings to reveal novel insights into how alpha rhythms organize spiking activity across visual and sensorimotor networks. Overall, this method enables powerful, large-scale electrophysiological experiments for the study of distributed neural computation.

Mixing novel and familiar cues modifies representations of familiar visual images and affects behavior.

While visual responses to familiar and novel stimuli have been extensively studied, it is unknown how neuronal representations of familiar stimuli are affected when they are interleaved with novel images. We examined a large-scale dataset from mice performing a visual go/no-go change detection task. After training with eight images, six novel images were interleaved with two familiar ones. Unexpectedly, we found that the behavioral performance in response to familiar images was impaired when they were mixed with novel images. When familiar images were interleaved with novel ones, the dimensionality of their representation increased, indicating a perturbation of their neuronal responses. Furthermore, responses to familiar images in the primary visual cortex were less predictive of responses in higher-order areas, indicating less efficient communication. Spontaneous correlations between neurons were predictive of responses to novel images, but less so to familiar ones. Our study demonstrates the modification of representations of familiar images by novelty.

Backward masking in mice requires visual cortex.

Visual masking can reveal the timescale of perception, but the underlying circuit mechanisms are not understood. Here we describe a backward masking task in mice and humans in which the location of a stimulus is potently masked. Humans report reduced subjective visibility that tracks behavioral deficits. In mice, both masking and optogenetic silencing of visual cortex (V1) reduce performance over a similar timecourse but have distinct effects on response rates and accuracy. Activity in V1 is consistent with masked behavior when quantified over long, but not short, time windows. A dual accumulator model recapitulates both mouse and human behavior. The model and subjects' performance imply that the initial spikes in V1 can trigger a correct response, but subsequent V1 activity degrades performance. Supporting this hypothesis, optogenetically suppressing mask-evoked activity in V1 fully restores accurate behavior. Together, these results demonstrate that mice, like humans, are susceptible to masking and that target and mask information is first confounded downstream of V1.

Coordinated changes in a cortical circuit sculpt effects of novelty on neural dynamics.

Recent studies have found dramatic cell-type specific responses to stimulus novelty, highlighting the importance of analyzing the cortical circuitry at the cell-type specific level of granularity to understand brain function. Although initial work classified and characterized activity for each cell type, the specific alterations in cortical circuitry-particularly when multiple novelty effects interact-remain unclear. To address this gap, we employed a large-scale public dataset of electrophysiological recordings in the visual cortex of awake, behaving mice using Neuropixels probes and designed population network models to investigate the observed changes in neural dynamics in response to a combination of distinct forms of novelty. The model parameters were rigorously constrained by publicly available structural datasets, including multi-patch synaptic physiology and electron microscopy data. Our systematic optimization approach identified tens of thousands of model parameter sets that replicate the observed neural activity. Analysis of these solutions revealed generally weaker connections under novel stimuli, as well as a shift in the balance e between SST and VIP populations. Along with this, PV and SST populations experienced overall more excitatory influences compared to excitatory and VIP populations. Our results also highlight the role of VIP neurons in multiple aspects of visual stimulus processing and altering gain and saturation dynamics under novel conditions. In sum, our findings provide a systematic characterization of how the cortical circuit adapts to stimulus novelty by combining multiple rich public datasets.

A survey of neurophysiological differentiation across mouse visual brain areas and timescales.

Neurophysiological differentiation (ND), a measure of the number of distinct activity states that a neural population visits over a time interval, has been used as a correlate of meaningfulness or subjective perception of visual stimuli. ND has largely been studied in non-invasive human whole-brain recordings where spatial resolution is limited. However, it is likely that perception is supported by discrete neuronal populations rather than the whole brain. Therefore, here we use Neuropixels recordings from the mouse brain to characterize the ND metric across a wide range of temporal scales, within neural populations recorded at single-cell resolution in localized regions. Using the spiking activity of thousands of simultaneously recorded neurons spanning 6 visual cortical areas and the visual thalamus, we show that the ND of stimulus-evoked activity of the entire visual cortex is higher for naturalistic stimuli relative to artificial ones. This finding holds in most individual areas throughout the visual hierarchy. Moreover, for animals performing an image change detection task, ND of the entire visual cortex (though not individual areas) is higher for successful detection compared to failed trials, consistent with the assumed perception of the stimulus. Together, these results suggest that ND computed on cellular-level neural recordings is a useful tool highlighting cell populations that may be involved in subjective perception.

Acute head-fixed recordings in awake mice with multiple Neuropixels probes.

Multi-electrode arrays such as Neuropixels probes enable electrophysiological recordings from large populations of single neurons with high temporal resolution. By using such probes, the activity from functionally interacting, yet distinct, brain regions can be measured simultaneously by inserting multiple probes into the same subject. However, the use of multiple probes in small animals such as mice requires the removal of a sizable fraction of the skull, while also minimizing tissue damage and keeping the brain stable during the recordings. Here, we describe a step-by-step process designed to facilitate reliable recordings from up to six Neuropixels probes simultaneously in awake, head-fixed mice. The procedure involves four stages: the implantation of a headframe and a removable glass coverslip, the precise positioning of the Neuropixels probes at targeted points on the brain surface, the placement of a perforated plastic imaging window and the insertion of the probes into the brain of an awake mouse. The approach provides access to multiple brain regions and has been successfully applied across hundreds of mice. The procedure has been optimized for dense recordings from the mouse visual system, but it can be adapted for alternative recording configurations to target multiple probes in other brain areas. The protocol is suitable for users with experience in stereotaxic surgery in mice.

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.

Higher-Order Thalamic Circuits Channel Parallel Streams of Visual Information in Mice.

Higher-order thalamic nuclei, such as the visual pulvinar, play essential roles in cortical function by connecting functionally related cortical and subcortical brain regions. A coherent framework describing pulvinar function remains elusive because of its anatomical complexity and involvement in diverse cognitive processes. We combined large-scale anatomical circuit mapping with high-density electrophysiological recordings to dissect a homolog of the pulvinar in mice, the lateral posterior thalamic nucleus (LP). We define three broad LP subregions based on correspondence between connectivity and functional properties. These subregions form corticothalamic loops biased toward ventral or dorsal stream cortical areas and contain separate representations of visual space. Silencing the visual cortex or superior colliculus revealed that they drive visual tuning properties in separate LP subregions. Thus, by specifying the driving input sources, functional properties, and downstream targets of LP circuits, our data provide a roadmap for understanding the mechanisms of higher-order thalamic function in vision.

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. Morphoelectrical properties of neurons measured on spatially dense electrode arrays have the potential to distinguish neuron types. We used high-density silicon 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 multichannel compared with single-channel waveforms. In visual cortex, unsupervised clustering identified the canonical regular-spiking (RS) and fast-spiking (FS) classes but also indicated 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. NEW & NOTEWORTHY It is challenging to identify neuron types with extracellular electrophysiology in vivo. We show that spatiotemporal action potentials measured on high-density electrode arrays can capture cell type-specific morphoelectrical properties, allowing classification of neurons across brain structures and within the cortex. Moreover, backpropagating action potentials are reliably detected in vivo from subpopulations of cortical and hippocampal neurons. Together, these results enhance the utility of dense extracellular electrophysiology for cell-type interrogation of brain network function.

Correlation of Synaptic Inputs in the Visual Cortex of Awake, Behaving Mice.

The subthreshold mechanisms that underlie neuronal correlations in awake animals are poorly understood. Here, we perform dual whole-cell recordings in the visual cortex (V1) of awake mice to investigate membrane potential (Vm) correlations between upper-layer sensory neurons. We find that the membrane potentials of neighboring neurons display large, correlated fluctuations during quiet wakefulness, including pairs of cells with disparate tuning properties. These fluctuations are driven by correlated barrages of excitation followed closely by inhibition (∼5-ms lag). During visual stimulation, low-frequency activity is diminished, and coherent high-frequency oscillations appear, even for non-preferred stimuli. These oscillations are generated by alternating excitatory and inhibitory inputs at a similar lag. The temporal sequence of depolarization for pairs of neurons is conserved during both spontaneous- and visually-evoked activity, suggesting a stereotyped flow of activation that may function to produce temporally precise "windows of opportunity" for additional synaptic inputs.

Feature-Specific Organization of Feedback Pathways in Mouse Visual Cortex.

Higher and lower cortical areas in the visual hierarchy are reciprocally connected [1]. Although much is known about how feedforward pathways shape receptive field properties of visual neurons, relatively little is known about the role of feedback pathways in visual processing. Feedback pathways are thought to carry top-down signals, including information about context (e.g., figure-ground segmentation and surround suppression) [2-5], and feedback has been demonstrated to sharpen orientation tuning of neurons in the primary visual cortex (V1) [6, 7]. However, the response characteristics of feedback neurons themselves and how feedback shapes V1 neurons' tuning for other features, such as spatial frequency (SF), remain largely unknown. Here, using a retrograde virus, targeted electrophysiological recordings, and optogenetic manipulations, we show that putatively feedback neurons in layer 5 (hereafter "L5 feedback") in higher visual areas, AL (anterolateral area) and PM (posteromedial area), display distinct visual properties in awake head-fixed mice. AL L5 feedback neurons prefer significantly lower SF (mean: 0.04 cycles per degree [cpd]) compared to PM L5 feedback neurons (0.15 cpd). Importantly, silencing AL L5 feedback reduced visual responses of V1 neurons preferring low SF (mean change in firing rate: -8.0%), whereas silencing PM L5 feedback suppressed responses of high-SF-preferring V1 neurons (-20.4%). These findings suggest that feedback connections from higher visual areas convey distinctly tuned visual inputs to V1 that serve to boost V1 neurons' responses to SF. Such like-to-like functional organization may represent an important feature of feedback pathways in sensory systems and in the nervous system in general.

Controlling brain states.

Neurons in mouse V1 increase their response to visual stimulation during locomotion. In this issue of Neuron, Lee et al. (2014) show that subthreshold optogenetic stimulation of a brainstem locomotion area can mimic the effect of locomotion on sensory processing.

Nicotinic modulation of cortical circuits.

The ascending cholinergic neuromodulatory system sends projections throughout cortex and has been shown to play an important role in a number of cognitive functions including arousal, working memory, and attention. However, despite a wealth of behavioral and anatomical data, understanding how cholinergic synapses modulate cortical function has been limited by the inability to selectively activate cholinergic axons. Now, with the development of optogenetic tools and cell-type specific Cre-driver mouse lines, it has become possible to stimulate cholinergic axons from the basal forebrain (BF) and probe cholinergic synapses in the cortex for the first time. Here we review recent work studying the cell-type specificity of nicotinic signaling in the cortex, synaptic mechanisms mediating cholinergic transmission, and the potential functional role of nicotinic modulation.

Subthreshold mechanisms underlying state-dependent modulation of visual responses.

The processing of sensory information varies widely across behavioral states. However, little is known about how behavioral states modulate the intracellular activity of cortical neurons to effect changes in sensory responses. Here, we performed whole-cell recordings from neurons in upper-layer primary visual cortex of awake mice during locomotion and quiet wakefulness. We found that the signal-to-noise ratio for sensory responses was improved during locomotion by two mechanisms: (1) a decrease in membrane potential variability leading to a reduction in background firing rates and (2) an enhancement in the amplitude and reliability of visually evoked subthreshold responses mediated by an increase in total conductance and a depolarization of the stimulus-evoked reversal potential. Consistent with the enhanced signal-to-noise ratio for visual responses during locomotion, we demonstrate that performance is improved in a visual detection task during this behavioral state.

Mechanisms generating dual-component nicotinic EPSCs in cortical interneurons.

Activation of cortical nicotinic receptors by cholinergic axons from the basal forebrain (BF) significantly impacts cortical function, and the loss of nicotinic receptors is a hallmark of aging and neurodegenerative disease. We have previously shown that stimulation of BF axons generates a fast α7 and a slow non-α7 receptor-dependent response in cortical interneurons. However, the synaptic mechanisms that underlie this dual-component nicotinic response remain unclear. Here, we report that fast α7 receptor-mediated EPSCs in the mouse cortex are highly variable and insensitive to perturbations of acetylcholinesterase (AChE), while slow non-α7 receptor-mediated EPSCs are reliable and highly sensitive to AChE activity. Based on these data, we propose that the fast and slow nicotinic responses reflect differences in synaptic structure between cholinergic varicosities activating α7 and non-α7 classes of nicotinic receptors.

Prolonged disynaptic inhibition in the cortex mediated by slow, non-α7 nicotinic excitation of a specific subset of cortical interneurons.

Cholinergic activation of nicotinic receptors in the cortex plays a critical role in arousal, attention, and learning. Here we demonstrate that cholinergic axons from the basal forebrain of mice excite a specific subset of cortical interneurons via a remarkably slow, non-α7 nicotinic receptor-mediated conductance. In turn, these inhibitory cells generate a delayed and prolonged wave of disynaptic inhibition in neighboring cortical neurons, altering the spatiotemporal pattern of inhibition in cortical circuits.