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Brain circuits are highly interconnected three-dimensional structures fabricated from components ranging vastly in size; from cell bodies to individual synapses. While neuronal activity can be visualized with advanced light microscopy (LM) techniques, the resolution of electron microscopy (EM) is critical for identifying synaptic connections between neurons. Here, we combine these two techniques, affording the advantage of each and allowing for measurements to be made of the same neural features across imaging platforms. We established an EM-label-free workflow utilizing inherent structural features to correlate in vivo two-photon LM and volumetric scanning EM (SEM) in the ferret visual cortex. By optimizing the volume SEM sample preparation protocol, imaging with the OnPoint detector, and utilizing the focal charge compensation device during serial block-face imaging, we achieved sufficient resolution and signal-to-noise ratio to analyze synaptic ultrastructure for hundreds of synapses within sample volumes. Our novel workflow provides a reliable method for quantitatively characterizing synaptic ultrastructure in functionally imaged neurons, providing new insights into neuronal circuit organization.

Establishing the fundamental principles that underlie the integration of excitatory and inhibitory presynaptic input populations is crucial to understanding how individual cortical neurons transform signals from peripheral receptors. Here we review recent studies using novel tools to examine the functional properties of excitatory synaptic inputs and the tuning of excitation and inhibition onto individual neurons. New evidence challenges existing synaptic connectivity rules and suggests a more complex functional synaptic architecture that supports a broad range of operations, enabling single neurons to encode multiple sensory features and flexibly shape their computations in the face of diverse sensory input., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

Circuits in the visual cortex integrate the information derived from separate ON (light-responsive) and OFF (dark-responsive) pathways to construct orderly columnar representations of stimulus orientation and visual space. How this transformation is achieved to meet the specific topographic constraints of each representation remains unclear. Here we report several novel features of ON-OFF convergence visualized by mapping the receptive fields of layer 2/3 neurons in the tree shrew (Tupaia belangeri) visual cortex using two-photon imaging of GCaMP6 calcium signals. We show that the spatially separate ON and OFF subfields of simple cells in layer 2/3 exhibit topologically distinct relationships with the maps of visual space and orientation preference. The centres of OFF subfields for neurons in a given region of cortex are confined to a compact region of visual space and display a smooth visuotopic progression. By contrast, the centres of the ON subfields are distributed over a wider region of visual space, display substantial visuotopic scatter, and have an orientation-specific displacement consistent with orientation preference map structure. As a result, cortical columns exhibit an invariant aggregate receptive field structure: an OFF-dominated central region flanked by ON-dominated subfields. This distinct arrangement of ON and OFF inputs enables continuity in the mapping of both orientation and visual space and the generation of a columnar map of absolute spatial phase.

A recent study shows conclusively that the koniocellular layers of the marmoset dorsal lateral geniculate nucleus have binocularly responsive neurons. This adds a new twist to the traditional view about binocular processing in the primate visual system and raises questions about the role of dorsal lateral geniculate nucleus in early binocular processing., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Two-photon (2P) imaging has proven to be a powerful tool for investigating neural structure and function both in brain slices and in intact systems. In vivo 2P imaging presents significant challenges in sample preparation, which are exacerbated in non-murine species. Here, we describe procedures for the effective virally mediated labeling of neurons and for the implantation of cranial windows for imaging. The procedures described here are applicable to a range of species, including mice, and are routinely used in ferrets and tree shrews to provide large-scale labeling of cortical volumes and high-quality imaging data.

The spatial arrangement of luminance increments (ON) and decrements (OFF) falling on the retina provides a wealth of information used by central visual pathways to construct coherent representations of visual scenes. But how the polarity of luminance change is represented in the activity of cortical circuits remains unclear. Using wide-field epifluorescence and two-photon imaging we demonstrate a robust modular representation of luminance polarity (ON or OFF) in the superficial layers of ferret primary visual cortex. Polarity-specific domains are found with both uniform changes in luminance and single light/dark edges, and include neurons selective for orientation and direction of motion. The integration of orientation and polarity preference is evident in the selectivity and discrimination capabilities of most layer 2/3 neurons. We conclude that polarity selectivity is an integral feature of layer 2/3 neurons, ensuring that the distinction between light and dark stimuli is available for further processing in downstream extrastriate areas., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Stimulus discrimination depends on the selectivity and variability of neural responses, as well as the size and correlation structure of the responsive population. For direction discrimination in visual cortex, only the selectivity of neurons has been well characterized across development. Here we show in ferrets that at eye opening, the cortical response to visual stimulation exhibits several immaturities, including a high density of active neurons that display prominent wave-like activity, a high degree of variability and strong noise correlations. Over the next three weeks, the population response becomes increasingly sparse, wave-like activity disappears, and variability and noise correlations are markedly reduced. Similar changes were observed in identified neuronal populations imaged repeatedly over days. Furthermore, experience with a moving stimulus was capable of driving a reduction in noise correlations over a matter of hours. These changes in variability and correlation contribute significantly to a marked improvement in direction discriminability over development.

To encode specific sensory inputs, cortical neurons must generate selective responses for distinct stimulus features. In principle, a variety of factors can contribute to the response selectivity of a cortical neuron: the tuning and strength of excitatory.sup.1-3 and inhibitory synaptic inputs.sup.4-6, dendritic nonlinearities.sup.7-9 and spike threshold.sup.10,11. Here we use a combination of techniques including in vivo whole-cell recording, synaptic- and cellular-resolution in vivo two-photon calcium imaging, and GABA ([gamma]-aminobutyric acid) neuron-selective optogenetic manipulation to dissect the factors that contribute to the direction-selective responses of layer 2/3 neurons in ferret visual cortex (V1). Two-photon calcium imaging of dendritic spines.sup.12,13 revealed that each neuron receives a mixture of excitatory synaptic inputs selective for the somatic preferred or null direction of motion. The relative number of preferred- and null-tuned excitatory inputs predicted a neuron's somatic direction preference, but failed to account for the degree of direction selectivity. By contrast, in vivo whole-cell patch-clamp recordings revealed a notable degree of direction selectivity in subthreshold responses that was significantly correlated with spiking direction selectivity. Subthreshold direction selectivity was predicted by the magnitude and variance of the response to the null direction of motion, and several lines of evidence, including conductance measurements, demonstrate that differential tuning of excitation and inhibition suppresses responses to the null direction of motion. Consistent with this idea, optogenetic inactivation of GABAergic neurons in layer 2/3 reduced direction selectivity by enhancing responses to the null direction. Furthermore, by optogenetically mapping connections of inhibitory neurons in layer 2/3 in vivo, we find that layer 2/3 inhibitory neurons make long-range, intercolumnar projections to excitatory neurons that prefer the opposite direction of motion. We conclude that intracortical inhibition exerts a major influence on the degree of direction selectivity in layer 2/3 of ferret V1 by suppressing responses to the null direction of motion.Inhibition to the null direction of motion has a critical role in the direction selectivity of neurons in ferret primary visual cortex., Author(s): Daniel E. Wilson [sup.1] [sup.2] [sup.3] [sup.4] , Benjamin Scholl [sup.1] , David Fitzpatrick [sup.1] Author Affiliations:(1) Max Planck Florida Institute for Neuroscience, Jupiter, USA(2) IMPRS for Brain and [...]

The visual cortex of carnivores and primates displays a high degree of modular network organization characterized by local clustering and structured long-range correlations of activity and functional properties. Excitatory networks display modular organization before the onset of sensory experience, but the developmental timeline for modular networks of GABAergic interneurons remains under-explored. Using in vivo calcium imaging of the ferret visual cortex, we find evidence that before visual experience, interneurons display weak orientation tuning and widespread, correlated activity in response to visual stimuli. Robust modular organization and orientation tuning are evident with as little as one week of visual experience. Furthermore, we find that the maturation of orientation tuning requires visual experience, while the reduction in widespread, correlated network activity does not. Thus, the maturation of inhibitory cortical networks occurs in a delayed, parallel process relative to excitatory neurons. Excitatory neurons in the carnivore and primate visual cortex display orientation selectivity arranged in a modular fashion before the onset of visual experience, but the developmental timeline for visual response selectivity of inhibitory neurons is unknown. Using in vivo calcium imaging in ferret visual cortex, the authors show that experience-dependent emergence of orientation selectivity develops in a modular fashion for inhibitory interneurons over the first week of visual experience. [ABSTRACT FROM AUTHOR]

In visual cortex, signals from the two eyes merge to form a coherent binocular representation. Here we investigate the synaptic interactions underlying the binocular representation of stimulus orientation in ferret visual cortex with in vivo calcium imaging of layer 2/3 neurons and their dendritic spines. Individual neurons with aligned somatic responses received a mixture of monocular and binocular synaptic inputs. Surprisingly, monocular pathways alone could not account for somatic alignment because ipsilateral monocular inputs poorly matched somatic preference. Binocular inputs exhibited different degrees of interocular alignment, and those with a high degree of alignment (congruent) had greater selectivity and somatic specificity. While congruent inputs were similar to others in measures of strength, simulations show that the number of active congruent inputs predicts aligned somatic output. Our study suggests that coherent binocular responses derive from connectivity biases that support functional amplification of aligned signals within a heterogeneous binocular intracortical network. [Display omitted] • Layer 2/3 neurons in visual cortex receive monocular and binocular synaptic inputs • Monocular inputs alone cannot account for somatic binocular alignment • Binocular congruent inputs exhibit functional properties ideally suited for alignment • Dominant influence of binocular congruent inputs derives from strength in numbers Scholl, Tepohl, et al. investigate how signals from the two eyes are combined within synaptic populations of individual neurons to shape a coherent representation. Rather than a convergence of monocular streams, they find that intracortical binocular networks are key to shaping alignment in cortical layer 2/3. [ABSTRACT FROM AUTHOR]

Understanding how cortical inhibition shapes circuit function requires identifying the connectivity rules relating the response properties of inhibitory interneurons and their postsynaptic targets. Here we explore the orientation tuning of layer 2/3 inhibitory inputs in the ferret visual cortex using a combination of in vivo axon imaging, functional input mapping, and physiology. Inhibitory boutons exhibit robust orientation-tuned responses with preferences that can differ significantly from the cortical column in which they reside. Inhibitory input fields measured with patterned optogenetic stimulation and intracellular recordings revealed that these inputs originate from a wide range of orientation domains, inconsistent with a model of co-tuned inhibition and excitation. Intracellular synaptic conductance measurements confirm that individual neurons can depart from a co-tuned regime. Our results argue against a simple rule for the arrangement of inhibitory inputs supplied by layer 2/3 circuits and suggest that heterogeneity in presynaptic inhibitory networks contributes to neural response properties. • GABAergic boutons have diverse orientation preferences within orientation domains • Single-neuron inhibitory input fields originate from diverse orientation domains • Conductance measurements show inhibition and excitation are not strictly co-tuned Scholl et al. examine the functional connectivity of layer 2/3 inhibitory inputs onto single neurons in the ferret visual cortex. This study argues against a simple rule describing the arrangement of inhibitory inputs supplied by layer 2/3 circuits. [ABSTRACT FROM AUTHOR]

Summary Functional circuits in the visual cortex require the coordinated activity of excitatory and inhibitory neurons. Molecular genetic approaches in the mouse have led to the “local non-specific pooling principle” of inhibitory connectivity, in which inhibitory neurons are untuned for stimulus features due to the random pooling of local inputs. However, it remains unclear whether this principle generalizes to species with a columnar organization of feature selectivity such as carnivores, primates, and humans. Here we use virally mediated GABAergic-specific GCaMP6f expression to demonstrate that inhibitory neurons in ferret visual cortex respond robustly and selectively to oriented stimuli. We find that the tuning of inhibitory neurons is inconsistent with the local non-specific pooling of excitatory inputs and that inhibitory neurons exhibit orientation-specific noise correlations with local and distant excitatory neurons. These findings challenge the generality of the non-specific pooling principle for inhibitory neurons, suggesting different rules for functional excitatory-inhibitory interactions in non-murine species. [ABSTRACT FROM AUTHOR]