The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo.

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.

Common modular architecture across diverse cortical areas in early development.

In order to deal with a complex environment, animals form a diverse range of neural representations that vary across cortical areas, ranging from largely unimodal sensory input to higher-order representations of goals, outcomes, and motivation. The developmental origin of this diversity is currently unclear, as representations could arise through processes that are already area-specific from the earliest developmental stages or alternatively, they could emerge from an initially common functional organization shared across areas. Here, we use spontaneous activity recorded with two-photon and widefield calcium imaging to reveal the functional organization across the early developing cortex in ferrets, a species with a well-characterized columnar organization and modular structure of spontaneous activity in the visual cortex. We find that in animals 7 to 14 d prior to eye-opening and ear canal opening, spontaneous activity in both sensory areas (auditory and somatosensory cortex, A1 and S1, respectively), and association areas (posterior parietal and prefrontal cortex, PPC and PFC, respectively) showed an organized and modular structure that is highly similar to the organization in V1. In all cortical areas, this modular activity was distributed across the cortical surface, forming functional networks that exhibit millimeter-scale correlations. Moreover, this modular structure was evident in highly coherent spontaneous activity at the cellular level, with strong correlations among local populations of neurons apparent in all cortical areas examined. Together, our results demonstrate a common distributed and modular organization across the cortex during early development, suggesting that diverse cortical representations develop initially according to similar design principles.

Self-organization of modular activity in immature cortical networks.

During development, cortical activity is organized into distributed modular patterns that are a precursor of the mature columnar functional architecture. Theoretically, such structured neural activity can emerge dynamically from local synaptic interactions through a recurrent network with effective local excitation with lateral inhibition (LE/LI) connectivity. Utilizing simultaneous widefield calcium imaging and optogenetics in juvenile ferret cortex prior to eye opening, we directly test several critical predictions of an LE/LI mechanism. We show that cortical networks transform uniform stimulations into diverse modular patterns exhibiting a characteristic spatial wavelength. Moreover, patterned optogenetic stimulation matching this wavelength selectively biases evoked activity patterns, while stimulation with varying wavelengths transforms activity towards this characteristic wavelength, revealing a dynamic compromise between input drive and the network's intrinsic tendency to organize activity. Furthermore, the structure of early spontaneous cortical activity - which is reflected in the developing representations of visual orientation - strongly overlaps that of uniform opto-evoked activity, suggesting a common underlying mechanism as a basis for the formation of orderly columnar maps underlying sensory representations in the brain.

Self-organization of modular activity in immature cortical networks.

During development, cortical activity is organized into distributed modular patterns that are a precursor of the mature columnar functional architecture. Theoretically, such structured neural activity can emerge dynamically from local synaptic interactions through a recurrent network with effective local excitation with lateral inhibition (LE/LI) connectivity. Utilizing simultaneous widefield calcium imaging and optogenetics in juvenile ferret cortex prior to eye opening, we directly test several critical predictions of an LE/LI mechanism. We show that cortical networks transform uniform stimulations into diverse modular patterns exhibiting a characteristic spatial wavelength. Moreover, patterned optogenetic stimulation matching this wavelength selectively biases evoked activity patterns, while stimulation with varying wavelengths transforms activity towards this characteristic wavelength, revealing a dynamic compromise between input drive and the network's intrinsic tendency to organize activity. Furthermore, the structure of early spontaneous cortical activity - which is reflected in the developing representations of visual orientation - strongly overlaps that of uniform opto-evoked activity, suggesting a common underlying mechanism as a basis for the formation of orderly columnar maps underlying sensory representations in the brain.

All-optical interrogation of millimeter-scale networks and application to developing ferret cortex.

BACKGROUND: Perception and behavior require coordinated activity of thousands of neurons operating in networks that span millimeters of brain area. In vivo calcium imaging approaches have proven exceptionally powerful for examining the structure of these networks at large scales, and optogenetics can allow for causal manipulations of large populations of neurons. However, realizing the full potential of these techniques requires the ability to simultaneously measure and manipulate distinct circuit elements on the scale of millimeters. NEW METHOD: We describe an opto-macroscope, an artifact-free, all-optical system capable of delivering patterned optogenetic stimulation with high spatial and temporal resolution across millimeters of brain while simultaneously imaging functional neural activity. RESULTS: We find that this approach provides direct manipulation of cortical regions ranging from hundreds of microns to several millimeters in area, allowing for the perturbation of individual brain areas or networks of functional domains. Using this system we find that spatially complex endogenous networks in the developing ferret visual cortex can be readily reactivated by precisely designed patterned optogenetic stimuli. COMPARISON WITH EXISTING METHODS: Our opto-macroscope extends current all-optical optogenetic approaches which operate on a cellular scale with multiphoton stimulation, and are poorly suited to investigate the millimeter-scale of many functional networks. It also builds upon other mesoscopic optogenetic techniques that lack simultaneous optical readouts of neural activity. CONCLUSIONS: The large-scale all-optical capabilities of our system make it a powerful new tool for investigating the contribution of cortical domains and brain areas to the functional neural networks that underlie perception and behavior.

Developmental maturation of millimeter-scale functional networks across brain areas.

Interacting with the environment to process sensory information, generate perceptions, and shape behavior engages neural networks in brain areas with highly varied representations, ranging from unimodal sensory cortices to higher-order association areas. Recent work suggests a much greater degree of commonality across areas, with distributed and modular networks present in both sensory and non-sensory areas during early development. However, it is currently unknown whether this initially common modular structure undergoes an equally common developmental trajectory, or whether such a modular functional organization persists in some areas-such as primary visual cortex-but not others. Here we examine the development of network organization across diverse cortical regions in ferrets of both sexes using in vivo widefield calcium imaging of spontaneous activity. We find that all regions examined, including both primary sensory cortices (visual, auditory, and somatosensory-V1, A1, and S1, respectively) and higher order association areas (prefrontal and posterior parietal cortices) exhibit a largely similar pattern of changes over an approximately 3 week developmental period spanning eye opening and the transition to predominantly externally-driven sensory activity. We find that both a modular functional organization and millimeter-scale correlated networks remain present across all cortical areas examined. These networks weakened over development in most cortical areas, but strengthened in V1. Overall, the conserved maintenance of modular organization across different cortical areas suggests a common pathway of network refinement, and suggests that a modular organization-known to encode functional representations in visual areas-may be similarly engaged in highly diverse brain areas.

Initial neighborhood biases and the quality of motion stimulation jointly influence the rapid emergence of direction preference in visual cortex.

Visual experience plays a critical role in the development of direction-selective responses in ferret visual cortex. In visually naive animals, presentation of a bidirectional "training" stimulus induces rapid increases in the direction-selective responses of single neurons that can be predicted by small but significant direction biases that are present in neighboring neurons at the onset of stimulation. In this study we used in vivo two-photon imaging of calcium signals to further explore the contribution of visual experience to the emergence of direction- selective responses in ferret visual cortex. The first set of experiments was designed to determine whether visual experience is required for the development of the initial neighborhood bias. In animals that were dark-reared until the time of eye opening, we found that individual neurons exhibited weak direction-selective responses accompanied by a reduced but statistically significant neighborhood bias, indicating that both features arise without the need for visual experience. The second set of experiments used a unidirectional training stimulus to assess the relative roles of the neighborhood bias and visual experience in determining the direction preference that cortical neurons acquire during direction training. We found that neurons became more responsive to the trained direction even when they were located in regions of the cortex with an initial neighborhood bias for the direction opposite the training stimulus. Together, these results suggest an adaptive developmental strategy for the elaboration of direction-selective responses, one in which experience-independent mechanisms provide a symmetry-breaking seed for the instructive effects of visual experience.

A redox-shifted fibroblast subpopulation emerges in the fibrotic lung.

Idiopathic pulmonary fibrosis (IPF) is an aggressive and thus far incurable disease, characterized by aberrant fibroblast-mediated extracellular matrix deposition. Our understanding of the disease etiology is incomplete; however, there is consensus that a reduction-oxidation (redox) imbalance plays a role. In this study we use the autofluorescent properties of two redox molecules, NAD(P)H and FAD, to quantify changes in their relative abundance in living lung tissue of mice with experimental lung fibrosis, and in freshly isolated cells from mouse lungs and humans with IPF. Our results identify cell population-specific intracellular redox changes in the lungs in experimental and human fibrosis. We focus particularly on redox changes within collagen producing cells, where we identified a bimodal distribution of NAD(P)H concentrations, establishing NAD(P)H high and NAD(P)H low sub-populations. NAD(P)H high fibroblasts exhibited elevated pro-fibrotic gene expression and decreased collagenolytic protease activity relative to NAD(P)H low fibroblasts. The NAD(P)H high population was present in healthy lungs but expanded with time after bleomycin injury suggesting a potential role in fibrosis progression. We identified a similar increased abundance of NAD(P)H high cells in freshly dissociated lungs of subjects with IPF relative to controls, and similar reductions in collagenolytic activity in this cell population. These data highlight the complexity of redox state changes in experimental and human pulmonary fibrosis and the need for selective approaches to restore redox imbalances in the fibrotic lung.

Essential role for a long-term depression mechanism in ocular dominance plasticity.

The classic example of experience-dependent cortical plasticity is the ocular dominance (OD) shift in visual cortex after monocular deprivation (MD). The experimental model of homosynaptic long-term depression (LTD) was originally introduced to study the mechanisms that could account for deprivation-induced loss of visual responsiveness. One established LTD mechanism is a loss of sensitivity to the neurotransmitter glutamate caused by internalization of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Although it has been shown that MD similarly causes a loss of AMPARs from visual cortical synapses, the contribution of this change to the OD shift has not been established. Using an herpes simplex virus (HSV) vector, we expressed in visual cortical neurons a peptide (G2CT) designed to block AMPAR internalization by hindering the association of the C-terminal tail of the AMPAR GluR2 subunit with the AP2 clathrin adaptor complex. We found that G2CT expression interferes with NMDA receptor (NMDAR)-dependent AMPAR endocytosis and LTD, without affecting baseline synaptic transmission. When expressed in vivo, G2CT completely blocked the OD shift and depression of deprived-eye responses after MD without affecting baseline visual responsiveness or experience-dependent response potentiation in layer 4 of visual cortex. These data suggest that AMPAR internalization is essential for the loss of synaptic strength caused by sensory deprivation in visual cortex.

Correction of fragile X syndrome in mice.

Fragile X syndrome (FXS) is the most common form of heritable mental retardation and the leading identified cause of autism. FXS is caused by transcriptional silencing of the FMR1 gene that encodes the fragile X mental retardation protein (FMRP), but the pathogenesis of the disease is unknown. According to one proposal, many psychiatric and neurological symptoms of FXS result from unchecked activation of mGluR5, a metabotropic glutamate receptor. To test this idea we generated Fmr1 mutant mice with a 50% reduction in mGluR5 expression and studied a range of phenotypes with relevance to the human disorder. Our results demonstrate that mGluR5 contributes significantly to the pathogenesis of the disease, a finding that has significant therapeutic implications for fragile X and related developmental disorders.

Visual processing: Systematic variation in light-dark bias across visual space.

Detecting changes in luminance is a fundamental property of the visual system. A new study shows that lights and darks are represented differently across visual space, with strong OFF bias in central vision and balanced ON/OFF in the periphery.

Tightly coupled inhibitory and excitatory functional networks in the developing primary visual cortex.

Intracortical inhibition plays a critical role in shaping activity patterns in the mature cortex. However, little is known about the structure of inhibition in early development prior to the onset of sensory experience, a time when spontaneous activity exhibits long-range correlations predictive of mature functional networks. Here, using calcium imaging of GABAergic neurons in the ferret visual cortex, we show that spontaneous activity in inhibitory neurons is already highly organized into distributed modular networks before visual experience. Inhibitory neurons exhibit spatially modular activity with long-range correlations and precise local organization that is in quantitative agreement with excitatory networks. Furthermore, excitatory and inhibitory networks are strongly co-aligned at both millimeter and cellular scales. These results demonstrate a remarkable degree of organization in inhibitory networks early in the developing cortex, providing support for computational models of self-organizing networks and suggesting a mechanism for the emergence of distributed functional networks during development.

Distributed network interactions and their emergence in developing neocortex.

The principles governing the functional organization and development of long-range network interactions in the neocortex remain poorly understood. Using in vivo widefield and two-photon calcium imaging of spontaneous activity patterns in mature ferret visual cortex, we find widespread modular correlation patterns that accurately predict the local structure of visually evoked orientation columns several millimeters away. Longitudinal imaging demonstrates that long-range spontaneous correlations are present early in cortical development before the elaboration of horizontal connections and predict mature network structure. Silencing feedforward drive through retinal or thalamic blockade does not eliminate early long-range correlated activity, suggesting a cortical origin. Circuit models containing only local, but heterogeneous, connections are sufficient to generate long-range correlated activity by confining activity patterns to a low-dimensional subspace via multisynaptic short-range interactions. These results suggest that local connections in early cortical circuits can generate structured long-range network correlations that guide the formation of visually evoked distributed functional networks.

Activation of NR2B-containing NMDA receptors is not required for NMDA receptor-dependent long-term depression.

The triggering of both NMDA receptor-dependent long-term potentiation (LTP) and long-term depression (LTD) in the CA1 region of the hippocampus requires a rise in postsynaptic calcium. A prominent hypothesis has been that the detailed properties of this postsynaptic calcium signal dictate whether LTP or LTD is generated by a given pattern of synaptic activity. Recently, however, evidence has been presented that the subunit composition of the NMDA receptor (NMDAR) determines whether a synapse undergoes LTP or LTD with NR2A-containing NMDARs triggering LTP and NR2B-containing NMDARs triggering LTD. In the present study, the role of NR2B-containing synaptic NMDARs in the induction of LTD in CA1 pyramidal cells has been studied using the selective NR2B antagonists, ifenprodil and Ro25-6981. While both antagonists reduced NMDAR-mediated synaptic currents, neither prevented induction of LTD. These results demonstrate that activation of NR2B-containing NMDARs is not an absolute requirement for the induction of LTD in the hippocampus.

GABAergic Neurons in Ferret Visual Cortex Participate in Functionally Specific Networks.

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.

Viral Injection and Cranial Window Implantation for In Vivo Two-Photon Imaging.

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.

A viral strategy for targeting and manipulating interneurons across vertebrate species.

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species, including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABAergic function in virtually any vertebrate species.

Modular Representation of Luminance Polarity in the Superficial Layers of Primary Visual Cortex.

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.

The development of cortical circuits for motion discrimination.

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.

Acute immunomodulatory effects of binge alcohol ingestion.

BACKGROUND: Blood alcohol is present in a third of trauma patients and has been associated with organ dysfunction. In both human studies and in animal models, it is clear that alcohol intoxication exerts immunomodulatory effects several hours to days after exposure, when blood alcohol is no longer detectable. The early immunomodulatory effects of alcohol while blood alcohol is still elevated are not well understood. METHODS: Human volunteers achieved binge alcohol intoxication after high-dose alcohol consumption. Blood was collected for analysis prior to alcohol ingestion, and 20 min, 2 h, and 5 h after alcohol ingestion. Flow cytometry was performed on isolated peripheral blood mononuclear cells, and cytokine generation in whole blood was measured by enzyme-linked immunosorbent assay (ELISA) after 24-h stimulation with lipopolysaccharide (LPS) and phytohemagglutinin-M (PHA) stimulation. RESULTS: An early pro-inflammatory state was evident at 20 min when blood alcohol levels were ∼130 mg/dL, which was characterized by an increase in total circulating leukocytes, monocytes, and natural killer cells. During this time, a transient increase in LPS-induced tumor necrosis factor (TNF)-α levels and enhanced LPS sensitivity occurred. At 2 and 5 h post-alcohol binge, an anti-inflammatory state was shown with reduced numbers of circulating monocytes and natural killer cells, attenuated LPS-induced interleukin (IL)-1ß levels, and a trend toward increased interleukin (IL)-10 levels. CONCLUSIONS: A single episode of binge alcohol intoxication exerted effects on the immune system that caused an early and transient pro-inflammatory state followed by an anti-inflammatory state.