Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex.

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types, of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1, AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex.

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently-developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1 AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

Primate V2 Receptive Fields Derived from Anatomically Identified Large-Scale V1 Inputs.

In the primate visual system, visual object recognition involves a series of cortical areas arranged hierarchically along the ventral visual pathway. As information flows through this hierarchy, neurons become progressively tuned to more complex image features. The circuit mechanisms and computations underlying the increasing complexity of these receptive fields (RFs) remain unidentified. To understand how this complexity emerges in the secondary visual area (V2), we investigated the functional organization of inputs from the primary visual cortex (V1) to V2 by combining retrograde anatomical tracing of these inputs with functional imaging of feature maps in macaque monkey V1 and V2. We found that V1 neurons sending inputs to single V2 orientation columns have a broad range of preferred orientations, but are strongly biased towards the orientation represented at the injected V2 site. For each V2 site, we then constructed a feedforward model based on the linear combination of its anatomically-identified large-scale V1 inputs, and studied the response proprieties of the generated V2 RFs. We found that V2 RFs derived from the linear feedforward model were either elongated versions of V1 filters or had spatially complex structures. These modeled RFs predicted V2 neuron responses to oriented grating stimuli with high accuracy. Remarkably, this simple model also explained the greater selectivity to naturalistic textures of V2 cells compared to their V1 input cells. Our results demonstrate that simple linear combinations of feedforward inputs can account for the orientation selectivity and texture sensitivity of V2 RFs.

Primate V2 Receptive Fields Derived from Anatomically Identified Large-Scale V1 Inputs.

In the primate visual system, visual object recognition involves a series of cortical areas arranged hierarchically along the ventral visual pathway. As information flows through this hierarchy, neurons become progressively tuned to more complex image features. The circuit mechanisms and computations underlying the increasing complexity of these receptive fields (RFs) remain unidentified. To understand how this complexity emerges in the secondary visual area (V2), we investigated the functional organization of inputs from the primary visual cortex (V1) to V2 by combining retrograde anatomical tracing of these inputs with functional imaging of feature maps in macaque monkey V1 and V2. We found that V1 neurons sending inputs to single V2 orientation columns have a broad range of preferred orientations, but are strongly biased towards the orientation represented at the injected V2 site. For each V2 site, we then constructed a feedforward model based on the linear combination of its anatomically-identified large-scale V1 inputs, and studied the response proprieties of the generated V2 RFs. We found that V2 RFs derived from the linear feedforward model were either elongated versions of V1 filters or had spatially complex structures. These modeled RFs predicted V2 neuron responses to oriented grating stimuli with high accuracy. Remarkably, this simple model also explained the greater selectivity to naturalistic textures of V2 cells compared to their V1 input cells. Our results demonstrate that simple linear combinations of feedforward inputs can account for the orientation selectivity and texture sensitivity of V2 RFs.

An optrode array for spatiotemporally-precise large-scale optogenetic stimulation of deep cortical layers in non-human primates.

Optogenetics has transformed studies of neural circuit function, but remains challenging to apply to non-human primates (NHPs). A major challenge is delivering intense, spatiotemporally-precise, patterned photostimulation across large volumes in deep tissue. Such stimulation is critical, for example, to modulate selectively deep-layer corticocortical feedback circuits. To address this need, we have developed the Utah Optrode Array (UOA), a 10×10 glass needle waveguide array fabricated atop a novel opaque optical interposer, and bonded to an electrically addressable µLED array. In vivo experiments with the UOA demonstrated large-scale, spatiotemporally precise, activation of deep circuits in NHP cortex. Specifically, the UOA permitted both focal (confined to single layers/columns), and widespread (multiple layers/columns) optogenetic activation of deep layer neurons, as assessed with multi-channel laminar electrode arrays, simply by varying the number of activated µLEDs and/or the irradiance. Thus, the UOA represents a powerful optoelectronic device for targeted manipulation of deep-layer circuits in NHP models.

An Optrode Array for Spatiotemporally Precise Large-Scale Optogenetic Stimulation of Deep Cortical Layers in Non-human Primates.

Optogenetics has transformed studies of neural circuit function, but remains challenging to apply in non-human primates (NHPs). A major challenge is delivering intense and spatially precise patterned photostimulation across large volumes in deep tissue. Here, we have developed and validated the Utah Optrode Array (UOA) to meet this critical need. The UOA is a 10×10 glass waveguide array bonded to an electrically-addressable µLED array. In vivo electrophysiology and immediate early gene (c-fos) immunohistochemistry demonstrated the UOA allows for large-scale spatiotemporally precise neuromodulation of deep tissue in macaque primary visual cortex. Specifically, the UOA permits both focal (single layers or columns), and large-scale (across multiple layers or columns) photostimulation of deep cortical layers, simply by varying the number of simultaneously activated µLEDs and/or the light irradiance. These results establish the UOA as a powerful tool for studying targeted neural populations within single or across multiple deep layers in complex NHP circuits.

A direct interareal feedback-to-feedforward circuit in primate visual cortex.

The mammalian sensory neocortex consists of hierarchically organized areas reciprocally connected via feedforward (FF) and feedback (FB) circuits. Several theories of hierarchical computation ascribe the bulk of the computational work of the cortex to looped FF-FB circuits between pairs of cortical areas. However, whether such corticocortical loops exist remains unclear. In higher mammals, individual FF-projection neurons send afferents almost exclusively to a single higher-level area. However, it is unclear whether FB-projection neurons show similar area-specificity, and whether they influence FF-projection neurons directly or indirectly. Using viral-mediated monosynaptic circuit tracing in macaque primary visual cortex (V1), we show that V1 neurons sending FF projections to area V2 receive monosynaptic FB inputs from V2, but not other V1-projecting areas. We also find monosynaptic FB-to-FB neuron contacts as a second motif of FB connectivity. Our results support the existence of FF-FB loops in primate cortex, and suggest that FB can rapidly and selectively influence the activity of incoming FF signals.

Stream-specific feedback inputs to the primate primary visual cortex.

The sensory neocortex consists of hierarchically-organized areas reciprocally connected via feedforward and feedback circuits. Feedforward connections shape the receptive field properties of neurons in higher areas within parallel streams specialized in processing specific stimulus attributes. Feedback connections have been implicated in top-down modulations, such as attention, prediction and sensory context. However, their computational role remains unknown, partly because we lack knowledge about rules of feedback connectivity to constrain models of feedback function. For example, it is unknown whether feedback connections maintain stream-specific segregation, or integrate information across parallel streams. Using viral-mediated labeling of feedback connections arising from specific cytochrome-oxidase stripes of macaque visual area V2, here we show that feedback to the primary visual cortex (V1) is organized into parallel streams resembling the reciprocal feedforward pathways. This suggests that functionally-specialized V2 feedback channels modulate V1 responses to specific stimulus attributes, an organizational principle potentially extending to feedback pathways in other sensory systems.

Improving the Usability of Virtual Reality Neuron Tracing with Topological Elements.

Researchers in the field of connectomics are working to reconstruct a map of neural connections in the brain in order to understand at a fundamental level how the brain processes information. Constructing this wiring diagram is done by tracing neurons through high-resolution image stacks acquired with fluorescence microscopy imaging techniques. While a large number of automatic tracing algorithms have been proposed, these frequently rely on local features in the data and fail on noisy data or ambiguous cases, requiring time-consuming manual correction. As a result, manual and semi-automatic tracing methods remain the state-of-the-art for creating accurate neuron reconstructions. We propose a new semi-automatic method that uses topological features to guide users in tracing neurons and integrate this method within a virtual reality (VR) framework previously used for manual tracing. Our approach augments both visualization and interaction with topological elements, allowing rapid understanding and tracing of complex morphologies. In our pilot study, neuroscientists demonstrated a strong preference for using our tool over prior approaches, reported less fatigue during tracing, and commended the ability to better understand possible paths and alternatives. Quantitative evaluation of the traces reveals that users' tracing speed increased, while retaining similar accuracy compared to a fully manual approach.

An Open Resource for Non-human Primate Optogenetics.

Optogenetics has revolutionized neuroscience in small laboratory animals, but its effect on animal models more closely related to humans, such as non-human primates (NHPs), has been mixed. To make evidence-based decisions in primate optogenetics, the scientific community would benefit from a centralized database listing all attempts, successful and unsuccessful, of using optogenetics in the primate brain. We contacted members of the community to ask for their contributions to an open science initiative. As of this writing, 45 laboratories around the world contributed more than 1,000 injection experiments, including precise details regarding their methods and outcomes. Of those entries, more than half had not been published. The resource is free for everyone to consult and contribute to on the Open Science Framework website. Here we review some of the insights from this initial release of the database and discuss methodological considerations to improve the success of optogenetic experiments in NHPs.

Top-down feedback controls spatial summation and response amplitude in primate visual cortex.

Sensory information travels along feedforward connections through a hierarchy of cortical areas, which, in turn, send feedback connections to lower-order areas. Feedback has been implicated in attention, expectation, and sensory context, but the mechanisms underlying these diverse feedback functions are unknown. Using specific optogenetic inactivation of feedback connections from the secondary visual area (V2), we show how feedback affects neural responses in the primate primary visual cortex (V1). Reducing feedback activity increases V1 cells' receptive field (RF) size, decreases their responses to stimuli confined to the RF, and increases their responses to stimuli extending into the proximal surround, therefore reducing surround suppression. Moreover, stronger reduction of V2 feedback activity leads to progressive increase in RF size and decrease in response amplitude, an effect predicted by a recurrent network model. Our results indicate that feedback modulates RF size, surround suppression and response amplitude, similar to the modulatory effects of visual spatial attention.

A Virtual Reality Visualization Tool for Neuron Tracing.

Tracing neurons in large-scale microscopy data is crucial to establishing a wiring diagram of the brain, which is needed to understand how neural circuits in the brain process information and generate behavior. Automatic techniques often fail for large and complex datasets, and connectomics researchers may spend weeks or months manually tracing neurons using 2D image stacks. We present a design study of a new virtual reality (VR) system, developed in collaboration with trained neuroanatomists, to trace neurons in microscope scans of the visual cortex of primates. We hypothesize that using consumer-grade VR technology to interact with neurons directly in 3D will help neuroscientists better resolve complex cases and enable them to trace neurons faster and with less physical and mental strain. We discuss both the design process and technical challenges in developing an interactive system to navigate and manipulate terabyte-sized image volumes in VR. Using a number of different datasets, we demonstrate that, compared to widely used commercial software, consumer-grade VR presents a promising alternative for scientists.

Circuits and Mechanisms for Surround Modulation in Visual Cortex.

Surround modulation (SM) is a fundamental property of sensory neurons in many species and sensory modalities. SM is the ability of stimuli in the surround of a neuron's receptive field (RF) to modulate (typically suppress) the neuron's response to stimuli simultaneously presented inside the RF, a property thought to underlie optimal coding of sensory information and important perceptual functions. Understanding the circuit and mechanisms for SM can reveal fundamental principles of computations in sensory cortices, from mouse to human. Current debate is centered over whether feedforward or intracortical circuits generate SM, and whether this results from increased inhibition or reduced excitation. Here we present a working hypothesis, based on theoretical and experimental evidence, that SM results from feedforward, horizontal, and feedback interactions with local recurrent connections, via synaptic mechanisms involving both increased inhibition and reduced recurrent excitation. In particular, strong and balanced recurrent excitatory and inhibitory circuits play a crucial role in the computation of SM.

Local Circuits of V1 Layer 4B Neurons Projecting to V2 Thick Stripes Define Distinct Cell Classes and Avoid Cytochrome Oxidase Blobs.

Decades of anatomical studies on the primate primary visual cortex (V1) have led to a detailed diagram of V1 intrinsic circuitry, but this diagram lacks information about the output targets of V1 cells. Understanding how V1 local processing relates to downstream processing requires identification of neuronal populations defined by their output targets. In primates, V1 layers (L)2/3 and 4B send segregated projections to distinct cytochrome oxidase (CO) stripes in area V2: neurons in CO blob columns project to thin stripes while neurons outside blob columns project to thick and pale stripes, suggesting functional specialization of V1-to-V2 CO streams. However, the conventional diagram of V1 shows all L4B neurons, regardless of their soma location in blob or interblob columns, as projecting selectively to CO blobs in L2/3, suggesting convergence of blob/interblob information in L2/3 blobs and, possibly, some V2 stripes. However, it is unclear whether all L4B projection neurons show similar local circuitries. Using viral-mediated circuit tracing, we have identified the local circuits of L4B neurons projecting to V2 thick stripes in macaque. Consistent with previous studies, we found the somata of this L4B subpopulation to reside predominantly outside blob columns; however, unlike previous descriptions of local L4B circuits, these cells consistently projected outside CO blob columns in all layers. Thus, the local circuits of these L4B output neurons, just like their extrinsic projections to V2, preserve CO streams. Moreover, the intra-V1 laminar patterns of axonal projections identify two distinct neuron classes within this L4B subpopulation, including a rare novel neuron type, suggestive of two functionally specialized output channels. SIGNIFICANCE STATEMENT: Conventional diagrams of primate primary visual cortex (V1) depict neuronal connections within and between different V1 layers, but lack information about the cells' downstream targets. This information is critical to understanding how local processing in V1 relates to downstream processing. We have identified the local circuits of a population of cells in V1 layer (L)4B that project to area V2. These cells' local circuits differ from classical descriptions of L4B circuits in both the laminar and functional compartments targeted by their axons, and identify two neuron classes. Our results demonstrate that both local intra-V1 and extrinsic V1-to-V2 connections of L4B neurons preserve CO-stream segregation, suggesting that across-stream integration occurs downstream of V1, and that output targets dictate local V1 circuitry.

ISAVS: Interactive Scalable Analysis and Visualization System.

Modern science is inundated with ever increasing data sizes as computational capabilities and image acquisition techniques continue to improve. For example, simulations are tackling ever larger domains with higher fidelity, and high-throughput microscopy techniques generate larger data that are fundamental to gather biologically and medically relevant insights. As the image sizes exceed memory, and even sometimes local disk space, each step in a scientific workflow is impacted. Current software solutions enable data exploration with limited interactivity for visualization and analytic tasks. Furthermore analysis on HPC systems often require complex hand-written parallel implementations of algorithms that suffer from poor portability and maintainability. We present a software infrastructure that simplifies end-to-end visualization and analysis of massive data. First, a hierarchical streaming data access layer enables interactive exploration of remote data, with fast data fetching to test analytics on subsets of the data. Second, a library simplifies the process of developing new analytics algorithms, allowing users to rapidly prototype new approaches and deploy them in an HPC setting. Third, a scalable runtime system automates mapping analysis algorithms to whatever computational hardware is available, reducing the complexity of developing scaling algorithms. We demonstrate the usability and performance of our system using a use case from neuroscience: filtering, registration, and visualization of tera-scale microscopy data. We evaluate the performance of our system using a leadership-class supercomputer, Shaheen II.

Corticocortical connection patterns reveal two distinct visual cortical areas bordering dorsal V2 in marmoset monkey.

The organization of the cortex located immediately anterior to the second visual area (V2), i.e., the third tier visual cortex, remains controversial, especially in New World primates. In particular, there is lack of consensus regarding the exact location and extent of the lower visual quadrant representation of the third visual area V3 (or ventrolateral posterior -VLP - of a different nomenclature). Microelectrode and connectional mapping studies have revealed the existence of an upper visual quadrant representation abutting dorsal V2 anteriorly, and bordered medially and laterally by representations of the lower visual quadrant. It remains unclear whether these lower field regions are both part of a single area V3, which is split into two patches by an interposed region of upper field representation, or whether they are the lower field representations of two different areas, the dorsomedial area (DM) and area V3/VLP, respectively. To address this question, we quantitatively analyzed the patterns of corticocortical afferent connections labeled by tracer injections targeted to these two lower field regions in the dorsal aspect of the third tier cortex. We found different inter-areal connectivity patterns arising from these two regions, strongly suggesting that they belong to two different visual areas. In particular, our results indicate that the dorsal aspect of the third tier cortex consists of two distinct areas: a full area DM, representing the lower quadrant medially, and the upper quadrant laterally, and the lower quadrant representation of V3/VLP, located laterally to upper field DM. DM is predominantly connected with areas of the dorsal visual stream, and V3/VLP with areas of the ventral stream. These results prompt further functional investigations of the third tier cortex, as previous studies of this cortical territory may have pooled response properties of two very different areas into a single area V3.

Two projection streams from macaque V1 to the pale cytochrome oxidase stripes of V2.

In the primate visual cortex, areas V1 and V2 distribute information they receive from the retina to virtually all extrastriate cortex, parsing this information into dorsal and ventral streams. Therefore, understanding the connectivity between V1 and V2 is crucial to understand visual cortical processing. Cytochrome oxidase staining in V2 reveals a repeating pattern of pale-thick-pale-thin stripes. V1 sends parallel output pathways to distinct V2 stripes. Previous models proposed either three or two parallel V1-to-V2 pathways in macaque, but both models viewed the two pale stripes within a single stripe cycle as a single compartment. However, recent studies have suggested that the two pale stripes may be functionally distinct, and in marmosets they also differ anatomically in the laminar origin of projections they receive from V1. Here we have asked whether the two pale stripes are also anatomically distinct in macaque. We made small retrograde tracer injections in different pale stripe types. We found that while both pale stripes receive a predominant V1 input from layers 2/3, only one set of pale stripes (pale lateral) receives significant projections from layer 4B, while the other set (pale medial) receives few or no layer 4B projections. Moreover, different tracer injections in nearby pale stripe types revealed that 97-99% of layer 2/3 cells only project to a single pale stripe type. These results demonstrate that in macaque, the two pale stripes are anatomically distinct compartments, and support the notion of two distinct projection streams from V1 to the two pale stripes of V2.

High-resolution mapping of anatomical connections in marmoset extrastriate cortex reveals a complete representation of the visual field bordering dorsal V2.

The primate visual cortex consists of many areas. The posterior areas (V1, V2, V3, and middle temporal) are thought to be common to all primate species. However, the organization of cortex immediately anterior to area V2 (the "third tier" cortex) remains controversial, particularly in New World primates. The main point of contention has been whether the third tier cortex consists of a single area V3, representing lower and upper visual quadrants in dorsal and ventral cortex, respectively, or of 2 distinct areas (the dorsomedial [DM] area and a V3-like area). Resolving this controversy is crucial to understand the function and evolution of the third tier cortex. We have addressed this issue in marmosets, by performing high-precision mapping of corticocortical connections in cortex bordering dorsal V2. Multiple closely spaced neuroanatomical tracer injections were placed across the full width of dorsal V2 or adjacent anterior cortex, and the location of resulting labeled cells mapped throughout whole flattened visual cortex. The resulting topographic patterns of labeled connections allowed us to define areas and their boundaries. We found that a complete representation of the visual field borders dorsal V2 and that the third tier cortex consists of 2 distinct areas. These results unequivocally support the DM model.

Four projection streams from primate V1 to the cytochrome oxidase stripes of V2.

In the primate visual system, areas V1 and V2 distribute information they receive from the retina to all higher cortical areas, sorting this information into dorsal and ventral streams. Therefore, knowledge of the organization of projections between V1 and V2 is crucial to understand how the cortex processes visual information. In primates, parallel output pathways from V1 project to distinct V2 stripes. The traditional tripartite division of V1-to-V2 projections was recently replaced by a bipartite scheme, in which thin stripes receive V1 inputs from blob columns, and thick and pale stripes receive common input from interblob columns. Here, we demonstrate that thick and pale stripes, instead, receive spatially segregated V1 inputs and that the interblob is partitioned into two compartments: the middle of the interblob projecting to pale stripes and the blob/interblob border region projecting to thick stripes. Double-labeling experiments further demonstrate that V1 cells project to either thick or pale stripes, but rarely to both. We also find laminar specialization of V1 outputs, with layer 4B contributing projections mainly to thick stripes, and no projections to one set of pale stripes. These laminar differences suggest different contribution of magno, parvo, and konio inputs to each V1 output pathway. These results provide a new foundation for parallel processing models of the visual system by demonstrating four V1-to-V2 pathways: blob columns-to-thin stripes, blob/interblob border columns-to-thick stripes, interblob columns-to-pale(lateral) stripes, layer 2/3-4A interblobs-to-pale(medial) stripes.

Anatomical evidence for classical and extra-classical receptive field completion across the discontinuous horizontal meridian representation of primate area V2.

In primates, a split of the horizontal meridian (HM) representation at the V2 rostral border divides this area into dorsal (V2d) and ventral (V2v) halves (representing lower and upper visual quadrants, respectively), causing retinotopically neighboring loci across the HM to be distant within V2. How is perceptual continuity maintained across this discontinuous HM representation? Injections of neuroanatomical tracers in marmoset V2d demonstrated that cells near the V2d rostral border can maintain retinotopic continuity within their classical and extra-classical receptive field (RF), by making both local and long-range intra- and interareal connections with ventral cortex representing the upper visual quadrant. V2d neurons located <0.9-1.3 mm from the V2d rostral border, whose RFs presumably do not cross the HM, make nonretinotopic horizontal connections with V2v neurons in the supra- and infragranular layers. V2d neurons located <0.6-0.9 mm from the border, whose RFs presumably cross the HM, in addition make retinotopic local connections with V2v neurons in layer 4. V2d neurons also make interareal connections with upper visual field regions of extrastriate cortex, but not of MT or MTc outside the foveal representation. Labeled connections in ventral cortex appear to represent the "missing" portion of the connectional fields in V2d across the HM. We conclude that connections between dorsal and ventral cortex can create visual field continuity within a second-order discontinuous visual topography.