Mesoscale functional connectivity in macaque visual areas.

Studies of resting-state functional connectivity (rsFC) have provided rich insights into the structures and functions of the human brain. However, most rsFC studies have focused on large-scale brain connectivity. To explore rsFC at a finer scale, we used intrinsic signal optical imaging to image the ongoing activity of the anesthetized macaque visual cortex. Differential signals from functional domains were used to quantify network-specific fluctuations. In 30-60 min resting-state imaging, a series of coherent activation patterns were observed in all three visual areas we examined (V1, V2, and V4). These patterns matched the known functional maps (ocular dominance, orientation, color) obtained in visual stimulation conditions. These functional connectivity (FC) networks fluctuated independently over time and exhibited similar temporal characteristics. Coherent fluctuations, however, were observed from orientation FC networks in different areas and even across two hemispheres. Thus, FC in the macaque visual cortex was fully mapped both on a fine scale and over a long range. Hemodynamic signals can be used to explore mesoscale rsFC in a submillimeter resolution.

Anatomy and function of an excitatory network in the visual cortex.

Circuits in the cerebral cortex consist of thousands of neurons connected by millions of synapses. A precise understanding of these local networks requires relating circuit activity with the underlying network structure. For pyramidal cells in superficial mouse visual cortex (V1), a consensus is emerging that neurons with similar visual response properties excite each other, but the anatomical basis of this recurrent synaptic network is unknown. Here we combined physiological imaging and large-scale electron microscopy to study an excitatory network in V1. We found that layer 2/3 neurons organized into subnetworks defined by anatomical connectivity, with more connections within than between groups. More specifically, we found that pyramidal neurons with similar orientation selectivity preferentially formed synapses with each other, despite the fact that axons and dendrites of all orientation selectivities pass near (<5 µm) each other with roughly equal probability. Therefore, we predict that mechanisms of functionally specific connectivity take place at the length scale of spines. Neurons with similar orientation tuning formed larger synapses, potentially enhancing the net effect of synaptic specificity. With the ability to study thousands of connections in a single circuit, functional connectomics is proving a powerful method to uncover the organizational logic of cortical networks.

3D Ultrastructure of Synaptic Inputs to Distinct GABAergic Neurons in the Mouse Primary Visual Cortex.

Synapses are the fundamental elements of the brain's complicated neural networks. Although the ultrastructure of synapses has been extensively studied, the difference in how synaptic inputs are organized onto distinct neuronal types is not yet fully understood. Here, we examined the cell-type-specific ultrastructure of proximal processes from the soma of parvalbumin-positive (PV+) and somatostatin-positive (SST+) GABAergic neurons in comparison with a pyramidal neuron in the mouse primary visual cortex (V1), using serial block-face scanning electron microscopy. Interestingly, each type of neuron organizes excitatory and inhibitory synapses in a unique way. First, we found that a subset of SST+ neurons are spiny, having spines on both soma and dendrites. Each of those spines has a highly complicated structure that has up to eight synaptic inputs. Next, the PV+ and SST+ neurons receive more robust excitatory inputs to their perisoma than does the pyramidal neuron. Notably, excitatory synapses on GABAergic neurons were often multiple-synapse boutons, making another synapse on distal dendrites. On the other hand, inhibitory synapses near the soma were often single-targeting multiple boutons. Collectively, our data demonstrate that synaptic inputs near the soma are differentially organized across cell types and form a network that balances inhibition and excitation in the V1.

Major Feedforward Thalamic Input Into Layer 4C of Primary Visual Cortex in Primate.

One of the underlying principles of how mammalian circuits are constructed is the relative influence of feedforward to recurrent synaptic drive. It has been dogma in sensory systems that the thalamic feedforward input is relatively weak and that there is a large amplification of the input signal by recurrent feedback. Here we show that in trichromatic primates there is a major feedforward input to layer 4C of primary visual cortex. Using a combination of 3D-electron-microscopy and 3D-confocal imaging of thalamic boutons we found that the average feedforward contribution was about 20% of the total excitatory input in the parvocellular (P) pathway, about 3 times the currently accepted values for primates. In the magnocellular (M) pathway it was around 15%, nearly twice the currently accepted values. New methods showed the total synaptic and cell densities were as much as 150% of currently accepted values. The new estimates of contributions of feedforward synaptic inputs into visual cortex call for a major revision of the design of the canonical cortical circuit.

Network anatomy and in vivo physiology of visual cortical neurons.

In the cerebral cortex, local circuits consist of tens of thousands of neurons, each of which makes thousands of synaptic connections. Perhaps the biggest impediment to understanding these networks is that we have no wiring diagrams of their interconnections. Even if we had a partial or complete wiring diagram, however, understanding the network would also require information about each neuron's function. Here we show that the relationship between structure and function can be studied in the cortex with a combination of in vivo physiology and network anatomy. We used two-photon calcium imaging to characterize a functional property--the preferred stimulus orientation--of a group of neurons in the mouse primary visual cortex. Large-scale electron microscopy of serial thin sections was then used to trace a portion of these neurons' local network. Consistent with a prediction from recent physiological experiments, inhibitory interneurons received convergent anatomical input from nearby excitatory neurons with a broad range of preferred orientations, although weak biases could not be rejected.

A Simple Network Architecture Accounts for Diverse Reward Time Responses in Primary Visual Cortex.

Many actions performed by animals and humans depend on an ability to learn, estimate, and produce temporal intervals of behavioral relevance. Exemplifying such learning of cued expectancies is the observation of reward-timing activity in the primary visual cortex (V1) of rodents, wherein neural responses to visual cues come to predict the time of future reward as behaviorally experienced in the past. These reward-timing responses exhibit significant heterogeneity in at least three qualitatively distinct classes: sustained increase or sustained decrease in firing rate until the time of expected reward, and a class of cells that reach a peak in firing at the expected delay. We elaborate upon our existing model by including inhibitory and excitatory units while imposing simple connectivity rules to demonstrate what role these inhibitory elements and the simple architectures play in sculpting the response dynamics of the network. We find that simply adding inhibition is not sufficient for obtaining the different distinct response classes, and that a broad distribution of inhibitory projections is necessary for obtaining peak-type responses. Furthermore, although changes in connection strength that modulate the effects of inhibition onto excitatory units have a strong impact on the firing rate profile of these peaked responses, the network exhibits robustness in its overall ability to predict the expected time of reward. Finally, we demonstrate how the magnitude of expected reward can be encoded at the expected delay in the network and how peaked responses express this reward expectancy. SIGNIFICANCE STATEMENT: Heterogeneity in single-neuron responses is a common feature of neuronal systems, although sometimes, in theoretical approaches, it is treated as a nuisance and seldom considered as conveying a different aspect of a signal. In this study, we focus on the heterogeneous responses in the primary visual cortex of rodents trained with a predictable delayed reward time. We describe under what conditions this heterogeneity can arise by self-organization, and what information it can convey. This study, while focusing on a specific system, provides insight onto how heterogeneity can arise in general while also shedding light onto mechanisms of reinforcement learning using realistic biological assumptions.

Activation of ß-adrenergic receptors in rat visual cortex expands astrocytic processes and reduces extracellular space volume.

Brain extracellular space (ECS) is an interconnected channel that allows diffusion-mediated transport of signaling molecules, metabolites, and drugs. We tested the hypothesis that ß-adrenergic receptor (ßAR) activation impacts extracellular diffusion-mediated transport of molecules through alterations in the morphology of astrocytes. Two structural parameters of ECS-volume fraction and tortuosity-govern extracellular diffusion. Volume fraction (α) is the volume of ECS relative to the total tissue volume. Tortuosity (λ) is a measure of the hindrance that molecules experience in the ECS, compared to a free medium. The real-time iontophoretic (RTI) method revealed that treatment of acutely prepared visual cortical slices of adult female rats with a ßAR agonist, DL-isoproterenol (ISO), decreases α significantly, from 0.22 ± 0.03 (mean ± SD) for controls without agonist to 0.18 ± 0.03 with ISO, without altering λ (control: 1.64 ± 0.04; ISO: 1.63 ± 0.04). Electron microscopy revealed that the ISO treatment significantly increased the cytoplasmic area of astrocytic distal endings per unit area of neuropil by 54%. These findings show that norepinephrine decreases α, in part, through an increase in astrocytic volume following ßAR activation. Norepinephrine is recognized to be released within the brain during the awake state and increase neurons' signal-to-noise ratio through modulation of neurons' biophysical properties. Our findings uncover a new mechanism for noradrenergic modulation of neuronal signals. Through astrocytic activation leading to a reduction of α, noradrenergic modulation increases extracellular concentration of neurotransmitters and neuromodulators, thereby facilitating neuronal interactions, especially during wakefulness. Synapse 70:307-316, 2016. © 2016 Wiley Periodicals, Inc.

The impairment of learning and memory and synaptic loss in mouse after chronic nitrite exposure.

The objective of this study is to understand the impairment of learning and memory in mouse after chronic nitrite exposure. The animal model of nitrite exposure in mouse was created with the daily intubation of nitrite in adult healthy male mice for 3 months. Furthermore, the mouse's learning and memory abilities were tested with Morris water maze, and the expression of Synaptophysin and γ-Synuclein was visualized with immunocytochemistry and Western blot. Our results showed that nitrite exposure significantly prolonged the escape latency period (ELP) and decreased the values of the frequency across platform (FAP) as well as the accumulative time in target quadrant (ATITQ) compared to control, in dose-dependent manner. In addition, after nitrite exposure, synaptophysin (SYN) positive buttons in the visual cortex was reduced, in contrast the increase of γ-synuclein positive cells. The results above were supported by Western blot as well. We conclude that nitrite exposure could lead to a decline in mice's learning and memory. The overexpression of γ-synuclein contributed to the synaptic loss, which is most likely the cause of learning and memory impairment. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1720-1730, 2016.

Deprivation-induced strengthening of presynaptic and postsynaptic inhibitory transmission in layer 4 of visual cortex during the critical period.

Inhibition from fast-spiking (FS) interneurons plays a crucial role in shaping cortical response properties and gating developmental periods of activity-dependent plasticity, yet the expression mechanisms underlying FS inhibitory plasticity remain largely unexplored. In layer 4 of visual cortex (V1), monocular deprivation (MD) induces either depression or potentiation of FS to star pyramidal neuron (FS→SP) synapses, depending on the age of onset (Maffei et al., 2004, 2006). This reversal in the sign (- to +) of plasticity occurs on the cusp of the canonical critical period (CP). To investigate the expression locus behind this switch in sign of inhibitory plasticity, mice underwent MD during the pre-CP [eye-opening to postnatal day (p)17] or CP (p22-p25), and FS→SP synaptic strength within layer 4 was assessed using confocal and immunoelectron microscopy, as well as optogenetic activation of FS cells to probe quantal amplitude at FS→SP synapses. Brief MD before p17 or p25 did not alter the density of FS→SP contacts. However, at the ultrastructural level, FS→SP synapses in deprived hemispheres during the CP, but not the pre-CP or in GAD65 knock-out mice, had larger synapses and increased docked vesicle density compared with synapses from the nondeprived control hemispheres. Moreover, FS→SP evoked miniature IPSCs increased in deprived hemispheres when MD was initiated during the CP, accompanied by an increase in the density of postsynaptic GABAA receptors at FS→SP synapses. These coordinated changes in FS→SP synaptic strength define an expression pathway modulating excitatory output during CP plasticity in visual cortex.

Imaging the Dynamics of Mammalian Neocortical Population Activity In-Vivo.

Neural computations underlying sensory perception, cognition and motor control are performed by populations of neurons at different anatomical and temporal scales. Few techniques are currently available for exploring dynamics of local and large range populations. Voltage-sensitive dye imaging (VSDI) reveals neural population activity in areas ranging from a few tens of microns to a couple of centimeters, or two areas up to ~10 cm apart. VSDI provides a sub-millisecond temporal resolution, and a spatial resolution of about 50 µm. The dye signal emphasizes subthreshold synaptic potentials. VSDI has been applied in the mouse, rat, gerbil, ferret, tree shrew, cat and monkey cortices, in order to explore lateral spread of retinotopic or somatotopic activation, the dynamic spatiotemporal pattern resulting from sensory activation, including the somatosensory, olfactory, auditory, and visual modalities, as well as motor preparation and the properties of spontaneously-occurring population activity. In this chapter we focus on VSDI in-vivo and review results obtained mostly in the visual system in our laboratory.

Nanoscopy of filamentous actin in cortical dendrites of a living mouse.

We demonstrate superresolution fluorescence microscopy (nanoscopy) of protein distributions in a mammalian brain in vivo. Stimulated emission depletion microscopy reveals the morphology of the filamentous actin in dendritic spines down to 40 µm in the molecular layer of the visual cortex of an anesthetized mouse. Consecutive recordings at 43-70 nm resolution reveal dynamical changes in spine morphology.

Ultrastructural differences impact cilia shape and external exposure across cell classes in the visual cortex.

A primary cilium is a membrane-bound extension from the cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. Primary cilia in the brain are less accessible than cilia on cultured cells or epithelial tissues because in the brain they protrude into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) but were absent from oligodendrocytes and microglia. Ultrastructural comparisons revealed that the base of the cilium and the microtubule organization differed between neurons and glia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting that cilia are poised to encounter locally released signaling molecules. Our analysis indicated that synapse proximity is likely due to random encounters in the neuropil, with no evidence that cilia modulate synapse activity as would be expected in tetrapartite synapses. The observed cell class differences in proximity to synapses were largely due to differences in external cilia length. Many key structural features that differed between neuronal and glial cilia influenced both cilium placement and shape and, thus, exposure to processes and synapses outside the cilium. Together, the ultrastructure both within and around neuronal and glial cilia suggest differences in cilia formation and function across cell types in the brain.

Liver production of sulfamidase reverses peripheral and ameliorates CNS pathology in mucopolysaccharidosis IIIA mice.

Mucopolysaccharidosis type IIIA (MPSIIIA) is an inherited lysosomal storage disease caused by deficiency of sulfamidase, resulting in accumulation of the glycosaminoglycan (GAG) heparan sulfate. It is characterized by severe progressive neurodegeneration, together with somatic alterations, which lead to death during adolescence. Here, we tested the ability of adeno-associated virus (AAV) vector-mediated genetic modification of either skeletal muscle or liver to revert the already established disease phenotype of 2-month-old MPSIIIA males and females. Intramuscular administration of AAV-Sulfamidase failed to achieve significant therapeutic benefit in either gender. In contrast, AAV8-mediated liver-directed gene transfer achieved high and sustained levels of circulating active sulfamidase, which reached normal levels in females and was fourfold higher in males, and completely corrected lysosomal GAG accumulation in most somatic tissues. Remarkably, a 50% reduction of GAG accumulation was achieved throughout the entire brain of males, which correlated with a partial improvement of the pathology of cerebellum and cortex. Liver-directed gene transfer expanded the lifespan of MPSIIIA males, underscoring the importance of reaching supraphysiological plasma levels of enzyme for maximal therapeutic benefit. These results show how liver-directed gene transfer can reverse somatic and ameliorate neurological pathology in MPSIIIA.

How thalamus connects to spiny stellate cells in the cat's visual cortex.

In the cat's visual cortex, the responses of simple cells seem to be totally determined by their thalamic input, yet only a few percent of the excitatory synapses in layer 4 arise from the thalamus. To resolve this discrepancy between structure and function, we used correlated light and electron microscopy to search individual spiny stellate cells (simple cells) for possible structural features that would explain the biophysical efficacy of the thalamic input, such as synaptic location on dendrites, size of postsynaptic densities, and postsynaptic targets. We find that thalamic axons form a small number of synapses with the spiny stellates (188 on average), that the median size of the synapses is slightly larger than that of other synapses on the dendrites of spiny stellates, that they are not located particularly proximal to the soma, and that they do not cluster on the dendrites. These findings point to alternative mechanisms, such as synchronous activation of the sparse thalamic synapses to boost the efficacy of the thalamic input. The results also support the idea that the thalamic input does not by itself determine the cortical response of spiny stellate cells, allowing the cortical microcircuit to amplify and modulate its response according to the particular context and computation being performed.

NrCAM deletion causes topographic mistargeting of thalamocortical axons to the visual cortex and disrupts visual acuity.

NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.

Gain modulation by nicotine in macaque v1.

Acetylcholine is a ubiquitous cortical neuromodulator implicated in cognition. In order to understand the potential for acetylcholine to play a role in visual attention, we studied nicotinic acetylcholine receptor (nAChR) localization and function in area V1 of the macaque. We found nAChRs presynaptically at thalamic synapses onto excitatory, but not inhibitory, neurons in the primary thalamorecipient layer 4c. Furthermore, consistent with the release enhancement suggested by this localization, we discovered that nicotine increases responsiveness and lowers contrast threshold in layer 4c neurons. We also found that nAChRs are expressed by GABAergic interneurons in V1 but rarely by pyramidal neurons, and that nicotine suppresses visual responses outside layer 4c. All sensory systems incorporate gain control mechanisms, or processes which dynamically alter input/output relationships. We demonstrate that at the site of thalamic input to visual cortex, the effect of this nAChR-mediated gain is an enhancement of the detection of visual stimuli.

The synaptic organization of the claustral projection to the cat's visual cortex.

The claustrum is a subcortical structure reciprocally connected with most areas of neocortex. This strategic location suggests an integrative role of the claustrum across different sensory modalities. However, our knowledge of the synaptic relationship between the neocortex and the claustrum is basic. In this study, we address this question through a structural investigation of the claustral projection to the ipsilateral primary visual cortex of the cat. Light microscopic reconstructions of axons from the entire thickness of cortex showed a very sparse innervation of the entire cortical depth, with most synaptic boutons in layers 2/3 and 6. Axons bearing numerous boutons terminaux and boutons en passant branched in these laminae. The sparse innervation did not seem to be compensated by particularly large synapses, given that the postsynaptic densities in the superficial layers are of comparable sizes (0.1 µm(2)) to other cortical synapses. All claustral synapses were asymmetric and in most cases targeted spines (87% in layer 4, 94% in layers 2/3 and 97% in layer 6). The pattern of innervation together with the known physiology of this projection suggests that the claustrum has a modulatory effect on visual cortex.

Pathway-specific utilization of synaptic zinc in the macaque ventral visual cortical areas.

Synaptic zinc is an activity-related neuromodulator, enriched in hippocampal mossy fibers and a subset of glutamatergic cortical projections, exclusive of thalamocortical or corticothalamic. Some degree of pathway specificity in the utilization of synaptic zinc has been reported in rodents. Here, we use focal injections of the retrograde tracer sodium selenite to identify zinc-positive (Zn+) projection neurons in the monkey ventral visual pathway. After injections in V1, V4, and TEO areas, neurons were detected preferentially in several feedback pathways but, unusually, were restricted to deeper layers without involvement of layers 2 or 3. Temporal injections resulted in more extensive labeling of both feedback and intratemporal association pathways. The Zn+ neurons had a broader laminar distribution, similar to results from standard retrograde tracers. After anterograde tracer injection in area posterior TE, electron microscopic analysis substantiated that a proportion of feedback synapses was co-labeled with zinc. Nearby injections, Zn+ intrinsic neurons concentrated in layer 2, but in temporal areas were also abundant in layer 6. These results indicate considerable pathway and laminar specificity as to which cortical neurons use synaptic zinc. Given the hypothesized roles of synaptic zinc, this is likely to result in distinct synaptic properties, possibly including differential synaptic plasticity within or across projections.

Structure and function of axo-axonic inhibition.

Inhibitory neurons in mammalian cortex exhibit diverse physiological, morphological, molecular, and connectivity signatures. While considerable work has measured the average connectivity of several interneuron classes, there remains a fundamental lack of understanding of the connectivity distribution of distinct inhibitory cell types with synaptic resolution, how it relates to properties of target cells, and how it affects function. Here, we used large-scale electron microscopy and functional imaging to address these questions for chandelier cells in layer 2/3 of the mouse visual cortex. With dense reconstructions from electron microscopy, we mapped the complete chandelier input onto 153 pyramidal neurons. We found that synapse number is highly variable across the population and is correlated with several structural features of the target neuron. This variability in the number of axo-axonic ChC synapses is higher than the variability seen in perisomatic inhibition. Biophysical simulations show that the observed pattern of axo-axonic inhibition is particularly effective in controlling excitatory output when excitation and inhibition are co-active. Finally, we measured chandelier cell activity in awake animals using a cell-type-specific calcium imaging approach and saw highly correlated activity across chandelier cells. In the same experiments, in vivo chandelier population activity correlated with pupil dilation, a proxy for arousal. Together, these results suggest that chandelier cells provide a circuit-wide signal whose strength is adjusted relative to the properties of target neurons.