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.

Responses to Pattern-Violating Visual Stimuli Evolve Differently Over Days in Somata and Distal Apical Dendrites.

Scientists have long conjectured that the neocortex learns patterns in sensory data to generate top-down predictions of upcoming stimuli. In line with this conjecture, different responses to pattern-matching vs pattern-violating visual stimuli have been observed in both spiking and somatic calcium imaging data. However, it remains unknown whether these pattern-violation signals are different between the distal apical dendrites, which are heavily targeted by top-down signals, and the somata, where bottom-up information is primarily integrated. Furthermore, it is unknown how responses to pattern-violating stimuli evolve over time as an animal gains more experience with them. Here, we address these unanswered questions by analyzing responses of individual somata and dendritic branches of layer 2/3 and layer 5 pyramidal neurons tracked over multiple days in primary visual cortex of awake, behaving female and male mice. We use sequences of Gabor patches with patterns in their orientations to create pattern-matching and pattern-violating stimuli, and two-photon calcium imaging to record neuronal responses. Many neurons in both layers show large differences between their responses to pattern-matching and pattern-violating stimuli. Interestingly, these responses evolve in opposite directions in the somata and distal apical dendrites, with somata becoming less sensitive to pattern-violating stimuli and distal apical dendrites more sensitive. These differences between the somata and distal apical dendrites may be important for hierarchical computation of sensory predictions and learning, since these two compartments tend to receive bottom-up and top-down information, respectively.

Hierarchical organization of cortical and thalamic connectivity.

The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource1, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.

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.

Differential encoding of temporal context and expectation under representational drift across hierarchically connected areas.

The classic view that neural populations in sensory cortices preferentially encode responses to incoming stimuli has been strongly challenged by recent experimental studies. Despite the fact that a large fraction of variance of visual responses in rodents can be attributed to behavioral state and movements, trial-history, and salience, the effects of contextual modulations and expectations on sensory-evoked responses in visual and association areas remain elusive. Here, we present a comprehensive experimental and theoretical study showing that hierarchically connected visual and association areas differentially encode the temporal context and expectation of naturalistic visual stimuli, consistent with the theory of hierarchical predictive coding. We measured neural responses to expected and unexpected sequences of natural scenes in the primary visual cortex (V1), the posterior medial higher order visual area (PM), and retrosplenial cortex (RSP) using 2-photon imaging in behaving mice collected through the Allen Institute Mindscope's OpenScope program. We found that information about image identity in neural population activity depended on the temporal context of transitions preceding each scene, and decreased along the hierarchy. Furthermore, our analyses revealed that the conjunctive encoding of temporal context and image identity was modulated by expectations of sequential events. In V1 and PM, we found enhanced and specific responses to unexpected oddball images, signaling stimulus-specific expectation violation. In contrast, in RSP the population response to oddball presentation recapitulated the missing expected image rather than the oddball image. These differential responses along the hierarchy are consistent with classic theories of hierarchical predictive coding whereby higher areas encode predictions and lower areas encode deviations from expectation. We further found evidence for drift in visual responses on the timescale of minutes. Although activity drift was present in all areas, population responses in V1 and PM, but not in RSP, maintained stable encoding of visual information and representational geometry. Instead we found that RSP drift was independent of stimulus information, suggesting a role in generating an internal model of the environment in the temporal domain. Overall, our results establish temporal context and expectation as substantial encoding dimensions in the visual cortex subject to fast representational drift and suggest that hierarchically connected areas instantiate a predictive coding mechanism.

Responses of pyramidal cell somata and apical dendrites in mouse visual cortex over multiple days.

The apical dendrites of pyramidal neurons in sensory cortex receive primarily top-down signals from associative and motor regions, while cell bodies and nearby dendrites are heavily targeted by locally recurrent or bottom-up inputs from the sensory periphery. Based on these differences, a number of theories in computational neuroscience postulate a unique role for apical dendrites in learning. However, due to technical challenges in data collection, little data is available for comparing the responses of apical dendrites to cell bodies over multiple days. Here we present a dataset collected through the Allen Institute Mindscope's OpenScope program that addresses this need. This dataset comprises high-quality two-photon calcium imaging from the apical dendrites and the cell bodies of visual cortical pyramidal neurons, acquired over multiple days in awake, behaving mice that were presented with visual stimuli. Many of the cell bodies and dendrite segments were tracked over days, enabling analyses of how their responses change over time. This dataset allows neuroscientists to explore the differences between apical and somatic processing and plasticity.

A whole-brain monosynaptic input connectome to neuron classes in mouse visual cortex.

Identification of structural connections between neurons is a prerequisite to understanding brain function. Here we developed a pipeline to systematically map brain-wide monosynaptic input connections to genetically defined neuronal populations using an optimized rabies tracing system. We used mouse visual cortex as the exemplar system and revealed quantitative target-specific, layer-specific and cell-class-specific differences in its presynaptic connectomes. The retrograde connectivity indicates the presence of ventral and dorsal visual streams and further reveals topographically organized and continuously varying subnetworks mediated by different higher visual areas. The visual cortex hierarchy can be derived from intracortical feedforward and feedback pathways mediated by upper-layer and lower-layer input neurons. We also identify a new role for layer 6 neurons in mediating reciprocal interhemispheric connections. This study expands our knowledge of the visual system connectomes and demonstrates that the pipeline can be scaled up to dissect connectivity of different cell populations across the mouse brain.

Measuring Stimulus-Evoked Neurophysiological Differentiation in Distinct Populations of Neurons in Mouse Visual Cortex.

Despite significant progress in understanding neural coding, it remains unclear how the coordinated activity of large populations of neurons relates to what an observer actually perceives. Since neurophysiological differences must underlie differences among percepts, differentiation analysis-quantifying distinct patterns of neurophysiological activity-has been proposed as an "inside-out" approach that addresses this question. This methodology contrasts with "outside-in" approaches such as feature tuning and decoding analyses, which are defined in terms of extrinsic experimental variables. Here, we used two-photon calcium imaging in mice of both sexes to systematically survey stimulus-evoked neurophysiological differentiation (ND) in excitatory neuronal populations in layers (L)2/3, L4, and L5 across five visual cortical areas (primary, lateromedial, anterolateral, posteromedial, and anteromedial) in response to naturalistic and phase-scrambled movie stimuli. We find that unscrambled stimuli evoke greater ND than scrambled stimuli specifically in L2/3 of the anterolateral and anteromedial areas, and that this effect is modulated by arousal state and locomotion. By contrast, decoding performance was far above chance and did not vary substantially across areas and layers. Differentiation also differed within the unscrambled stimulus set, suggesting that differentiation analysis may be used to probe the ethological relevance of individual stimuli.

VIP interneurons in mouse primary visual cortex selectively enhance responses to weak but specific stimuli.

Vasoactive intestinal peptide-expressing (VIP) interneurons in the cortex regulate feedback inhibition of pyramidal neurons through suppression of somatostatin-expressing (SST) interneurons and, reciprocally, SST neurons inhibit VIP neurons. Although VIP neuron activity in the primary visual cortex (V1) of mouse is highly correlated with locomotion, the relevance of locomotion-related VIP neuron activity to visual coding is not known. Here we show that VIP neurons in mouse V1 respond strongly to low contrast front-to-back motion that is congruent with self-motion during locomotion but are suppressed by other directions and contrasts. VIP and SST neurons have complementary contrast tuning. Layer 2/3 contains a substantially larger population of low contrast preferring pyramidal neurons than deeper layers, and layer 2/3 (but not deeper layer) pyramidal neurons show bias for front-to-back motion specifically at low contrast. Network modeling indicates that VIP-SST mutual antagonism regulates the gain of the cortex to achieve sensitivity to specific weak stimuli without compromising network stability.

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.

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.

Hiding Within the Cracks: Case Report of Rare Sutural Bone Found at the Nasion.

Pathology such as skull fractures can be misdiagnosed in the presence of anatomical variations. One variant that has had little description in the literature are the sutural bones associated with the nasal bones. Herein, we describe a case of a rare sutural bone at the nasion, between the bones of the right nasal, frontal, and maxillary frontal process. To our knowledge, this is the first report of such a variant bone in this location, and such it should be considered by clinicians when evaluating patients for pathology in this region.

Manipulation of GABA in the ventral pallidum, but not the nucleus accumbens, induces intense, preferential, fat consumption in rats.

Injections of the GABAA antagonist bicuculline into the medial ventral pallidum (VPm) induce marked increases in food intake, but nothing is known about the way in which these injections alter the distribution of intake in a macronutrient selection situation. We investigated this topic by adapting rats to a diet containing independent sources of protein, carbohydrate and fat, and then examining the effects of intra-VPm bicuculline on diet selection. Under these conditions, bicuculline produced a massive, preferential increase in fat intake with subjects consuming a mean of 97% of their calories from fat. Furthermore, all treated subjects ate fat before any other macronutrient, suggesting that the animals' behavior was directed selectively toward this dietary component even before consumption had begun. Similar effects were not observed following food deprivation, which exerted its largest effect on carbohydrate intake. To compare the intra-VPm bicuculline response to that seen after activation of GABA receptors in the nucleus accumbens shell (AcbSh), a major source of projections to the VPm, we conducted similar experiments with intra-AcbSh injections of muscimol and baclofen. These injections also enhanced food intake, but did not reproduce the selective preference for fat seen after intra-VPm bicuculline. These experiments provide the first demonstration of preferential enhancement of fat intake following manipulations of a nonpeptide neurotransmitter. Since mean intakes of fat under baseline conditions and after deprivation tended to be lower than those of carbohydrates, it seems unlikely that the effects of intra-VPm bicuculline are related to the intrinsic "rewarding" properties of fat, but might rather reflect the induction of a state of "fat craving."