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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.

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Neurophysiological recording with a new probe often yields better signal quality than with a used probe. Why does the signal quality degrade after only a few experiments? Here, we considered silicon probes in which the contacts are densely packed, and each contact is coated with a conductive polymer that increases its surface area. We tested 12 Cambridge Neurotech silicon probes during 61 recording sessions from the brain of three marmosets. Out of the box, each probe arrived with an electrodeposited polymer coating on 64 gold contacts and an impedance of around 50 kΩ. With repeated use, the impedance increased and there was a corresponding decrease in the number of well-isolated neurons. Imaging of the probes suggested that the reduction in signal quality was due to a gradual loss of the polymer coating. To rejuvenate the probes, we first stripped the contacts, completely removing their polymer coating, and then recoated them in a solution of 10 mM 3,4-Ethylenedioxythiophene (EDOT) monomer with 11 mM Poly(sodium 4-styrenesulfonate) (PSS) using a current density of about 3 mA/cm2 for 30 s. This recoating process not only returned probe impedance to around 50 kΩ but also yielded significantly improved signal quality during neurophysiological recordings. Thus, insertion into the brain promoted the loss of the polymer that coated the contacts of the silicon probes. This led to degradation of signal quality, but recoating rejuvenated the probes.NEW & NOTEWORTHY With repeated use, a silicon probe's ability to isolate neurons degrades. As a result, the probe is often discarded after only a handful of uses. Here, we demonstrate a major source of this problem and then produce a solution to rejuvenate the probes.

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In vivo recordings in freely behaving animals are crucial to understand the neuronal circuit basis of behavior. Although current multi-channel silicon probes provide unparalleled sampling density, the study of interacting neuronal populations requires the implantation of multiple probes across different regions of the brain. Ideally, these probes should be independently adjustable, to maximize the yield, and recoverable, to mitigate costs. In this work, we describe the implementation of a miniaturized 3D-printed headgear system for chronic in vivo recordings in mice using independently movable silicon probes targeting multiple brain regions. We successfully demonstrated the performance of the headgear by simultaneously recording the neuronal activity in the prelimbic cortex and dorsal hippocampus. The system proved to be sturdy, ensuring high-quality stable recordings and permitted reuse of the silicon probes, with no observable interference in mouse innate behaviors.

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To understand the neural mechanisms of behavior, it is necessary to both monitor and perturb the activity of ensembles of neurons with high specificity. While neural ensemble recordings have been available for decades, progress in high-resolution manipulation techniques has lagged behind. Optogenetics has enabled the manipulation of genetically defined cell types in behaving animals, and recent developments, including multipoint nanofabricated light sources, provide spatiotemporal resolution on a par with that of physiological recordings. Here we review current advances in optogenetic methods for cellular-resolution stimulation and intervention, as well as their integration with real-time neural recordings for closed-loop experimentation. We discuss how these approaches open the door to new kinds of experiments aimed at dissecting the role of specific neural patterns and discrete cellular populations in orchestrating the activity of brain circuits that support behavior and cognition.

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Neurons in the visual system can be spatially organized according to their response properties such as receptive field location and feature selectivity. For example, the visual cortex of many mammalian species contains orientation and direction columns where neurons with similar preferences are clustered. Here, we examine whether such a columnar structure exists in the mouse superior colliculus (SC), a prominent visual center for motion processing. By performing large-scale physiological recording and two-photon calcium imaging in adult male and female mice, we show that direction-selective neurons in the mouse SC are not organized into stereotypical columns as a function of their preferred directions, although clusters of similarly tuned neurons are seen in a minority of mice. Nearby neurons can prefer similar or opposite directions in a largely position-independent manner. This finding holds true regardless of animal state (anesthetized vs awake, running vs stationary), SC depth (most superficial lamina vs deeper in the SC), research technique (calcium imaging vs electrophysiology), and stimulus type (drifting gratings vs moving dots, full field vs small patch). Together, these results challenge recent reports of region-specific organizations in the mouse SC and reveal how motion direction is represented in this important visual center.

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In recent years, the advent of the so-called silicon probes has made it possible to homogeneously sample spikes and local field potentials (LFPs) from a regular grid of cortical recording sites. In principle, this allows inferring the laminar location of the sites based on the spatiotemporal pattern of LFPs recorded along the probe, as in the well-known current source-density (CSD) analysis. This approach, however, has several limitations, since it relies on visual identification of landmark features (i.e., current sinks and sources) by human operators - features that can be absent from the CSD pattern if the probe does not span the whole cortical thickness, thus making manual labelling harder. Furthermore, as any manual annotation procedure, the typical CSD-based workflow for laminar identification of recording sites is affected by subjective judgment undermining the consistency and reproducibility of results. To overcome these limitations, we developed an alternative approach, based on finding the optimal match between the LFPs recorded along a probe in a given experiment and a template LFP profile that was computed using 18 recording sessions, in which the depth of the recording sites had been recovered through histology. We show that this method can achieve an accuracy of 79 µm in recovering the cortical depth of recording sites and a 76% accuracy in inferring their laminar location. As such, our approach provides an alternative to CSD that, being fully automated, is less prone to the idiosyncrasies of subjective judgment and works reliably also for recordings spanning a limited cortical stretch.

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Visual systems have evolved to recognize and extract features from complex scenes using limited sensory information. Contour perception is essential to this process and can occur despite breaks in the continuity of neighboring features. Such robustness of the animal visual system to degraded or occluded shapes may also give rise to an interesting phenomenon of optical illusions. These illusions provide a great opportunity to decipher neural computations underlying contour integration and object detection. Kanizsa illusory contours have been shown to evoke responses in the early visual cortex despite the lack of direct receptive field activation. Recurrent processing between visual areas has been proposed to be involved in this process. However, it is unclear whether higher visual areas directly contribute to the generation of illusory responses in the early visual cortex. Using behavior, in vivo electrophysiology, and optogenetics, we first show that the primary visual cortex (V1) of male mice responds to Kanizsa illusory contours. Responses to Kanizsa illusions emerge later than the responses to the contrast-defined real contours in V1. Second, we demonstrate that illusory responses are orientation-selective. Finally, we show that top-down feedback controls the neural correlates of illusory contour perception in V1. Our results suggest that higher-order visual areas may fill in the missing information in the early visual cortex necessary for illusory contour perception.SIGNIFICANCE STATEMENT Perception of the Kanizsa illusory contours is impaired in neurodevelopmental disorders such as schizophrenia, autism, and Williams syndrome. However, the mechanism of the illusory contour perception is poorly understood. Here we describe the behavioral and neural correlates of Kanizsa illusory contours perception in mice, a genetically tractable model system. We show that top-down feedback controls the neural responses to Kanizsa illusion in V1. To our knowledge, this is the first description of the neural correlates of the Kanizsa illusion in mice and the first causal demonstration of their regulation by top-down feedback.

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Decoding laminar information across deep brain structures and cortical regions is necessary in order to understand the neuronal ensembles that represent cognition and memory. Large animal models are essential for translational research due to their gyrencephalic neuroanatomy and significant white matter composition. A lack of long-length probes with appropriate stiffness allowing penetration to deeper structures with minimal damage to the neural interface is one of the major technical limitations to applying the approaches currently utilized in lower order animals to large animals. We therefore tested the performance of multichannel silicon probes of various solutions and designs that were developed specifically for large animal electrophysiology. Neurophysiological signals from dorsal hippocampus were recorded in chronically implanted awake behaving Yucatan pigs. Single units and local field potentials were analyzed to evaluate performance of given silicon probes over time. EDGE-style probes had the highest yields during intra-hippocampal recordings in pigs, making them the most suitable for chronic implantations and awake behavioral experimentation. In addition, the cross-sectional area of silicon probes was found to be a crucial determinant of silicon probe performance over time, potentially due to reduction of damage to the neural interface. Novel 64-channel EDGE-style probes tested acutely produced an optimal single unit separation and a denser sampling of the laminar structure, identifying these research silicon probes as potential candidates for chronic implantations. This study provides an analysis of multichannel silicon probes designed for large animal electrophysiology of deep laminar brain structures, and suggests that current designs are reaching the physical thresholds necessary for long-term (∼1 month) recordings with single-unit resolution.

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The ability to flexibly navigate an environment relies on a hippocampal-dependent cognitive map. External space can be internally mapped at different spatial resolutions. However, whether hippocampal spatial coding resolution can rapidly adapt to local features of an environment remains unclear. To explore this possibility, we recorded the firing of hippocampal neurons in mice navigating virtual reality environments, embedding or not local visual cues (virtual 3D objects) in specific locations. Virtual objects enhanced spatial coding resolution in their vicinity with a higher proportion of place cells, smaller place fields, increased spatial selectivity and stability. This effect was highly dynamic upon objects manipulations. Objects also improved temporal coding resolution through improved theta phase precession and theta timescale spike coordination. We propose that the fast adaptation of hippocampal spatial coding resolution to local features of an environment could be relevant for large-scale navigation.

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The hippocampus is integral to working and episodic memory and is a central region of interest in diseases affecting these processes. Pig models are widely used in translational research and may provide an excellent bridge between rodents and nonhuman primates for CNS disease models because of their gyrencephalic neuroanatomy and significant white matter composition. However, the laminar structure of the pig hippocampus has not been well characterized. Therefore, we histologically characterized the dorsal hippocampus of Yucatan miniature pigs and quantified the cytoarchitecture of the hippocampal layers. We then utilized stereotaxis combined with single-unit electrophysiological mapping to precisely place multichannel laminar silicon probes into the dorsal hippocampus without the need for image guidance. We used in vivo electrophysiological recordings of simultaneous laminar field potentials and single-unit activity in multiple layers of the dorsal hippocampus to physiologically identify and quantify these layers under anesthesia. Consistent with previous reports, we found the porcine hippocampus to have the expected archicortical laminar structure, with some anatomical and histological features comparable to the rodent and others to the primate hippocampus. Importantly, we found these distinct features to be reflected in the laminar electrophysiology. This characterization, as well as our electrophysiology-based methodology targeting the porcine hippocampal lamina combined with high-channel-count silicon probes, will allow for analysis of spike-field interactions during normal and disease states in both anesthetized and future awake behaving neurophysiology in this large animal.

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Familiarity of the environment changes the way we perceive and encode incoming information. However, the neural substrates underlying this phenomenon are poorly understood. Here we describe a new form of experience-dependent low-frequency oscillations in the primary visual cortex (V1) of awake adult male mice. The oscillations emerged in visually evoked potentials and single-unit activity following repeated visual stimulation. The oscillations were sensitive to the spatial frequency content of a visual stimulus and required the mAChRs for their induction and expression. Finally, ongoing visually evoked θ (4-8 Hz) oscillations boost the visually evoked potential amplitude of incoming visual stimuli if the stimuli are presented at the high excitability phase of the oscillations. Our results demonstrate that an oscillatory code can be used to encode familiarity and serves as a gate for oncoming sensory inputs.SIGNIFICANCE STATEMENT Previous experience can influence the processing of incoming sensory information by the brain and alter perception. However, the mechanistic understanding of how this process takes place is lacking. We have discovered that persistent low-frequency oscillations in the primary visual cortex encode information about familiarity and the spatial frequency of the stimulus. These familiarity evoked oscillations influence neuronal responses to the oncoming stimuli in a way that depends on the oscillation phase. Our work demonstrates a new mechanism of visual stimulus feature detection and learning.

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Intracortical microstimulation (ICMS) is one of the most widely employed techniques for providing causal evidence of the relationship between neuronal activity and specific motor, perceptual, or even cognitive functions. In recent years, several new types of linear multielectrode silicon probes have been developed, allowing researchers to sample neuronal activity at different depths along the same cortical site simultaneously and with high spatial precision. Nevertheless, silicon multielectrode probes have been rarely employed for ICMS studies and, more importantly, it is unknown whether and to what extent they can be used for combined recording and stimulation experiments. Here, we addressed these issues during both acute and chronic conditions. First, we compared the behavioral outcomes of ICMS delivered to the hand region of a monkey's motor cortex with multielectrode silicon probes, commercially available multisite stainless-steel probes and single-tip glass-coated tungsten microelectrodes. The results for all three of the probes were reliable and similar. Furthermore, we tested the impact of long-train ICMS delivered through chronically implanted silicon probes at different time intervals, from 1 to 198 days after ICMS sessions, showing that although the number of recorded neurons decreased over time, in line with previous studies, ICMS did not alter silicon probes' recording capabilities. These findings indicate that in ICMS experiments, the performance of linear multielectrode silicon probes is comparable to that of both single-tip and multielectrode stainless-steel probes, suggesting that the silicon probes can be successfully used for combined recording and stimulation studies in chronic conditions.

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Electrophysiological data acquisition systems introduce various distortions into the signals they record. While such distortions were discussed previously, their effects are often not appreciated. Here I show that the biphasic shape of cortical spike-triggered LFP average (stLFP), reported in multiple studies, is likely an artefact introduced by high-pass filter of the neural data acquisition system when the actual stLFP has a single trough around the zero lag.

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Recent reports in human demonstrate a role of theta-gamma coupling in memory for spatial episodes and a lack of coupling in people experiencing temporal lobe epilepsy, but the mechanisms are unknown. Using multisite silicon probe recordings of epileptic rats engaged in episodic-like object recognition tasks, we sought to evaluate the role of theta-gamma coupling in the absence of epileptiform activities. Our data reveal a specific association between theta-gamma (30-60 Hz) coupling at the proximal stratum radiatum of CA1 and spatial memory deficits. We targeted the microcircuit mechanisms with a novel approach to identify putative interneuronal types in tetrode recordings (parvalbumin basket cells in particular) and validated classification criteria in the epileptic context with neurochemical identification of intracellularly recorded cells. In epileptic rats, putative parvalbumin basket cells fired poorly modulated at the falling theta phase, consistent with weaker inputs from Schaffer collaterals and attenuated gamma oscillations, as evaluated by theta-phase decomposition of current-source density signals. We propose that theta-gamma interneuronal rhythmopathies of the temporal lobe are intimately related to episodic memory dysfunction in this condition.

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The coordinated activity of neural ensembles across multiple interconnected regions has been challenging to study in the mammalian brain with cellular resolution using conventional recording tools. For instance, neural systems regulating learned behaviors often encompass multiple distinct structures that span the brain. To address this challenge we developed a three-dimensional (3D) silicon microprobe capable of simultaneously measuring extracellular spike and local field potential activity from 1,024 electrodes. The microprobe geometry can be precisely configured during assembly to target virtually any combination of four spatially distinct neuroanatomical planes. Here we report on the operation of such a device built for high-throughput monitoring of neural signals in the orbitofrontal cortex and several nuclei in the basal ganglia. We perform analysis on systems-level dynamics and correlations during periods of conditioned behavioral responding and rest, demonstrating the technology's ability to reveal functional organization at multiple scales in parallel in the mouse brain.

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Identifying the properties of correlations in the firing of neocortical neurons is central to our understanding of cortical information processing. It has been generally assumed, by virtue of the columnar organization of the neocortex, that the firing of neurons residing in a certain vertical domain is highly correlated. On the other hand, firing correlations between neurons steeply decline with horizontal distance. Technical difficulties in sampling neurons with sufficient spatial information have precluded the critical evaluation of these notions. We used 128-channel "silicon probes" to examine the spike-count noise correlations during spontaneous activity between multiple neurons with identified laminar position and over large horizontal distances in the anesthetized rat barrel cortex. Eigen decomposition of correlation coefficient matrices revealed that the laminar position of a neuron is a significant determinant of these correlations, such that the fluctuations of layer 5B/6 neurons are in opposite direction to those of layers 5A and 4. Moreover, we found that within each experiment, the distribution of horizontal, intralaminar spike-count correlation coefficients, up to a distance of ∼1.5 mm, is practically identical to the distribution of vertical correlations. Taken together, these data reveal that the neuron's laminar position crucially affects its role in cortical processing. Moreover, our analyses reveal that this laminar effect extends over several functional columns. We propose that within the cortex the influence of the horizontal elements exists in a dynamic balance with the influence of the vertical domain and this balance is modulated with brain states to shape the network's behavior.

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Coherent neuronal activity in the hippocampal-entorhinal circuit is a critical mechanism for episodic memory function, which is typically impaired in temporal lobe epilepsy. To better understand how this mechanism is implemented and degraded in this condition, we used normal and epileptic rats to examine theta activity accompanying active exploration. Assisted by multisite recordings of local field potentials (LFPs) and layer-specific profiling of input pathways, we provide detailed quantification of the proximodistal coherence of theta activity in the dorsal hippocampus of these animals. Normal rats showed stronger coordination between the temporoammonic and perforant entorhinal inputs (measured from lamina-specific current source density signals) at proximal locations, i.e., closer to CA3; while epileptic rats exhibited stronger interactions at distal locations, i.e., closer to subiculum. This opposing trend in epileptic rats was associated with the reorganization of the temporoammonic and perforant pathways that accompany hippocampal sclerosis, the pathological hallmark of this disease. In addition to this connectivity constraint, we discovered that the appropriate timing between entorhinal inputs arriving over several theta cycles at the proximal and distal ends of the dorsal hippocampus was impaired in epileptic rats. Computational reconstruction of LFP signals predicted that restoring timing variability has a major impact on repairing theta coherence. This manipulation, when tested pharmacologically via systemic administration of group III mGluR antagonists, successfully re-established theta coordination of LFPs in epileptic rats. Thus, proximodistal organization of entorhinal inputs is instrumental in temporal lobe physiology and a candidate mechanism to study cognitive comorbidities of temporal lobe epilepsy.

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