Dendritic spikes enhance stimulus selectivity in cortical neurons in vivo.

Neuronal dendrites are electrically excitable: they can generate regenerative events such as dendritic spikes in response to sufficiently strong synaptic input. Although such events have been observed in many neuronal types, it is not well understood how active dendrites contribute to the tuning of neuronal output in vivo. Here we show that dendritic spikes increase the selectivity of neuronal responses to the orientation of a visual stimulus (orientation tuning). We performed direct patch-clamp recordings from the dendrites of pyramidal neurons in the primary visual cortex of lightly anaesthetized and awake mice, during sensory processing. Visual stimulation triggered regenerative local dendritic spikes that were distinct from back-propagating action potentials. These events were orientation tuned and were suppressed by either hyperpolarization of membrane potential or intracellular blockade of NMDA (N-methyl-d-aspartate) receptors. Both of these manipulations also decreased the selectivity of subthreshold orientation tuning measured at the soma, thus linking dendritic regenerative events to somatic orientation tuning. Together, our results suggest that dendritic spikes that are triggered by visual input contribute to a fundamental cortical computation: enhancing orientation selectivity in the visual cortex. Thus, dendritic excitability is an essential component of behaviourally relevant computations in neurons.

Maternal Loss of Ube3a Impairs Experience-Driven Dendritic Spine Maintenance in the Developing Visual Cortex.

UNLABELLED: Dendritic spines are a morphological feature of the majority of excitatory synapses in the mammalian neocortex and are motile structures with shapes and lifetimes that change throughout development. Proper cortical development and function, including cortical contributions to learning and memory formation, require appropriate experience-dependent dendritic spine remodeling. Dendritic spine abnormalities have been reported for many neurodevelopmental disorders, including Angelman syndrome (AS), which is caused by the loss of the maternally inherited UBE3A allele (encoding ubiquitin protein ligase E3A). Prior studies revealed that UBE3A protein loss leads to reductions in dendritic spine density and diminished excitatory synaptic transmission. However, the decrease in spine density could come from either a reduction in spine formation or an increase in spine elimination. Here, we used acute and longitudinal in vivo two-photon microscopy to investigate developmental and experience-dependent changes in the numbers, dynamics, and morphology of layer 5 pyramidal neuron apical dendritic spines in the primary visual cortex of control and AS model mice (Ube3a(m-/p+) mice). We found that neurons in AS model mice undergo a greater elimination of dendritic spines than wild-type mice during the end of the first postnatal month. However, when raised in darkness, spine density and dynamics were indistinguishable between control and AS model mice, which indicates that decreased spine density in AS model mice reflects impaired experience-driven spine maintenance. Our data thus demonstrate an experience-dependent anatomical substrate by which the loss of UBE3A reduces dendritic spine density and disrupts cortical circuitry. SIGNIFICANCE STATEMENT: Reduced dendritic spine densities are common in the neurodevelopmental disorder Angelman syndrome (AS). Because prior reports were based on postmortem tissue, it was unknown whether this anatomical deficit arises from decreased spine formation and/or increased spine elimination. Here, we used in vivo two-photon imaging to track spines over multiple days in a mouse model of AS. We found that spine formation is normal, but experience-dependent spine maintenance is reduced in the visual cortex of AS model mice. Our data pinpoint the anatomical process underlying the loss of dendritic spines, which can account for the decreased excitatory synaptic connectivity associated with AS. Therefore, normalizing spine maintenance is a potential therapeutic strategy.

Ube3a loss increases excitability and blunts orientation tuning in the visual cortex of Angelman syndrome model mice.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of the maternally inherited allele of UBE3AUbe3aSTOP/p+ mice recapitulate major features of AS in humans and allow conditional reinstatement of maternal Ube3a with the expression of Cre recombinase. We have recently shown that AS model mice exhibit reduced inhibitory drive onto layer (L)2/3 pyramidal neurons of visual cortex, which contributes to a synaptic excitatory/inhibitory imbalance. However, it remains unclear how this loss of inhibitory drive affects neural circuits in vivo. Here we examined visual cortical response properties in individual neurons to explore the consequences of Ube3a loss on intact cortical circuits and processing. Using in vivo patch-clamp electrophysiology, we measured the visually evoked responses to square-wave drifting gratings in L2/3 regular-spiking (RS) neurons in control mice, Ube3a-deficient mice, and mice in which Ube3a was conditionally reinstated in GABAergic neurons. We found that Ube3a-deficient mice exhibited enhanced pyramidal neuron excitability in vivo as well as weaker orientation tuning. These observations are the first to show alterations in cortical computation in an AS model, and they suggest a basis for cortical dysfunction in AS.NEW & NOTEWORTHY Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of the gene UBE3A Using electrophysiological recording in vivo, we describe visual cortical dysfunctions in a mouse model of AS. Aberrant cellular properties in AS model mice could be improved by reinstating Ube3a in inhibitory neurons. These findings suggest that inhibitory neurons play a substantial role in the pathogenesis of AS.

Target-specific effects of somatostatin-expressing interneurons on neocortical visual processing.

A diverse array of interneuron types regulates activity in the mammalian neocortex. Two of the most abundant are the fast-spiking, parvalbumin-positive (PV(+)) interneurons, which target the axosomatic region of pyramidal cells, and the somatostatin-positive (SOM(+)) interneurons, which target the dendrites. Recent work has focused on the influence of PV(+) and SOM(+) interneurons on pyramidal cells. However, the connections among PV(+) and SOM(+) interneurons are poorly understood and could play an important role in cortical circuitry, since their interactions may alter the net influence on pyramidal cell output. We used an optogenetic approach to investigate the effect of SOM(+) interneurons on pyramidal cells and PV(+) interneurons during visual stimulation in mouse primary visual cortex. We find that SOM(+) interneuron activation suppresses PV(+) cell spiking at least twice as potently as pyramidal cell spiking during visual stimulation. This differential effect of SOM(+) cell stimulation is detectable even when only two to three SOM(+) cells are activated. Importantly, the remaining responses to oriented gratings in PV(+) cells are more orientation tuned and temporally modulated, suggesting that SOM(+) activity unmasks this tuning by suppressing untuned input. Our results highlight the importance of SOM(+) inhibition of PV(+) interneurons during sensory processing. This prominent competitive inhibition between interneuron types leads to a reconfiguration of inhibition along the somatodendritic axis of pyramidal cells, and enhances the orientation selectivity of PV(+) cells.

Selective representations of texture and motion in mouse higher visual areas.

The mouse visual cortex contains interconnected higher visual areas, but their functional specializations are unclear. Here, we used a data-driven approach to examine the representations of complex visual stimuli by L2/3 neurons across mouse higher visual areas, measured using large-field-of-view two-photon calcium imaging. Using specialized stimuli, we found higher fidelity representations of texture in area LM, compared to area AL. Complementarily, we found higher fidelity representations of motion in area AL, compared to area LM. We also observed this segregation of information in response to naturalistic videos. Finally, we explored how receptive field models of visual cortical neurons could produce the segregated representations of texture and motion we observed. These selective representations could aid in behaviors such as visually guided navigation.

Experience-dependent binocular competition in the visual cortex begins at eye opening.

Visual experience begins at eye opening, but current models consider cortical circuitry to be resistant to experience-dependent competitive modification until the activation of a later critical period. Here we examine this idea using optical imaging to map the time course of receptive field refinement in normal mice, mice in which the contralateral eye never opens and mice in which the contralateral eye is silenced. We found that the refinement of ipsilateral eye retinotopy is retarded by contralateral deprivation, but accelerated by silencing of the contralateral eye. Patterned visual experience through the ipsilateral eye is required for this acceleration. These differences are most obvious at postnatal day 15, long before the start of the critical period, indicating that experience-dependent binocular plasticity occurs much earlier than was previously thought. Furthermore, these results suggest that the quality of activity, in terms of signal to noise, and not the quantity, determines robust receptive field development.

Diesel2p mesoscope with dual independent scan engines for flexible capture of dynamics in distributed neural circuitry.

Imaging the activity of neurons that are widely distributed across brain regions deep in scattering tissue at high speed remains challenging. Here, we introduce an open-source system with Dual Independent Enhanced Scan Engines for Large field-of-view Two-Photon imaging (Diesel2p). Combining optical design, adaptive optics, and temporal multiplexing, the system offers subcellular resolution over a large field-of-view of ~25 mm2, encompassing distances up to 7 mm, with independent scan engines. We demonstrate the flexibility and various use cases of this system for calcium imaging of neurons in the living brain.

Flexible simultaneous mesoscale two-photon imaging of neural activity at high speeds.

Understanding brain function requires monitoring local and global brain dynamics. Two-photon imaging of the brain across mesoscopic scales has presented trade-offs between imaging area and acquisition speed. We describe a flexible cellular resolution two-photon microscope capable of simultaneous video rate acquisition of four independently targetable brain regions spanning an approximate five-millimeter field of view. With this system, we demonstrate the ability to measure calcium activity across mouse sensorimotor cortex at behaviorally relevant timescales.

Deficits in higher visual area representations in a mouse model of Angelman syndrome.

BACKGROUND: Sensory processing deficits are common in individuals with neurodevelopmental disorders. One hypothesis is that deficits may be more detectable in downstream, "higher" sensory areas. A mouse model of Angelman syndrome (AS), which lacks expression of the maternally inherited Ube3a allele, has deficits in synaptic function and experience-dependent plasticity in the primary visual cortex. Thus, we hypothesized that AS model mice have deficits in visually driven neuronal responsiveness in downstream higher visual areas (HVAs). METHODS: Here, we used intrinsic signal optical imaging and two-photon calcium imaging to map visually evoked neuronal activity in the primary visual cortex and HVAs in response to an array of stimuli. RESULTS: We found a highly specific deficit in HVAs. Drifting gratings that changed speed caused a strong response in HVAs in wildtype mice, but this was not observed in littermate AS model mice. Further investigation with two-photon calcium imaging revealed the effect to be largely driven by aberrant responses of inhibitory interneurons, suggesting a cellular basis for higher level, stimulus-selective cortical dysfunction in AS. CONCLUSION: Assaying downstream, or "higher" circuitry may provide a more sensitive measure for circuit dysfunction in mouse models of neurodevelopmental disorders. TRIAL REGISTRATION: Not applicable.

Introduction to the Biophotonics Congress 2018 feature issue.

The guest editors introduce a feature issue containing papers based on research presented at the OSA Biophotonics Congress (the former BIOMED) held in Hollywood, FL, 2-6 April, 2018.

Mice use robust and common strategies to discriminate natural scenes.

Mice use vision to navigate and avoid predators in natural environments. However, their visual systems are compact compared to other mammals, and it is unclear how well mice can discriminate ethologically relevant scenes. Here, we examined natural scene discrimination in mice using an automated touch-screen system. We estimated the discrimination difficulty using the computational metric structural similarity (SSIM), and constructed psychometric curves. However, the performance of each mouse was better predicted by the mean performance of other mice than SSIM. This high inter-mouse agreement indicates that mice use common and robust strategies to discriminate natural scenes. We tested several other image metrics to find an alternative to SSIM for predicting discrimination performance. We found that a simple, primary visual cortex (V1)-inspired model predicted mouse performance with fidelity approaching the inter-mouse agreement. The model involved convolving the images with Gabor filters, and its performance varied with the orientation of the Gabor filter. This orientation dependence was driven by the stimuli, rather than an innate biological feature. Together, these results indicate that mice are adept at discriminating natural scenes, and their performance is well predicted by simple models of V1 processing.

Genotype- and sex-dependent effects of altered Cntnap2 expression on the function of visual cortical areas.

BACKGROUND: Autism spectrum disorder (ASD) is a heritable, heterogeneous neurodevelopmental disorder that is four times more likely to affect males than females. Despite this overt sex bias, it is unclear how genetic mutations associated with ASD alter cortical circuitry to produce the behavioral phenotypes by which ASD is diagnosed. Contactin-associated protein-like 2 (CNTNAP2) is an ASD-associated gene, and while Cntnap2 knockout (KO) mice recapitulate many of the features of ASD, the effect on cortical circuitry is poorly understood. Moreover, although heterozygous (Het) mice are the more relevant genotype for ASD-linked CNTNAP2 mutations in humans, to our knowledge, no effects in Het mice have been previously reported. METHODS: Intrinsic signal optical imaging was used to measure functional visual responses in primary and higher visual cortical areas in male and female Cntnap2 KO, Het, and wild-type (WT) mice. Main effect of genotype was assessed with one-way ANOVA. Visual responses were also measured in P17-18 and P30-32 KO and WT mice. Main effects of age and genotype were assessed using two-way ANOVA. RESULTS: Visually evoked activity in dorsal stream associated higher visual areas in both KO and Het adult males was decreased relative to WT adult males. This decrease was not observed in adult females. Additionally, no significant difference was observed between WT and KO males at P17-18 with differences beginning to emerge at P30-32. CONCLUSIONS: The functional responses of cortical circuitry in male mice are more strongly affected by Cntnap2 mutations than females, an effect present even in Hets. The observed differences in males emerge with development beginning at P30-32. These results reveal genotype- and sex-dependent effects of altered Cntnap2 expression and can shed light on the sex-dependent incidence of ASD.

Stream-dependent development of higher visual cortical areas.

Multiple cortical areas contribute to visual processing in mice. However, the functional organization and development of higher visual areas are unclear. Here we used intrinsic signal optical imaging and two-photon calcium imaging to map visual responses in adult and developing mice. We found that visually driven activity was well correlated among higher visual areas within two distinct subnetworks resembling the dorsal and ventral visual streams. Visual response magnitude in dorsal stream areas slowly increased over the first 2 weeks of visual experience. By contrast, ventral stream areas exhibited strong responses shortly after eye opening. Neurons in a dorsal stream area showed little change in their tuning sharpness to oriented gratings while those in a ventral stream area increased stimulus selectivity and expanded their receptive fields significantly. Together, these findings provide a functional basis for grouping subnetworks of mouse visual areas and revealed stream differences in the development of receptive field properties.

Technologies for imaging neural activity in large volumes.

Neural circuitry has evolved to form distributed networks that act dynamically across large volumes. Conventional microscopy collects data from individual planes and cannot sample circuitry across large volumes at the temporal resolution relevant to neural circuit function and behaviors. Here we review emerging technologies for rapid volume imaging of neural circuitry. We focus on two critical challenges: the inertia of optical systems, which limits image speed, and aberrations, which restrict the image volume. Optical sampling time must be long enough to ensure high-fidelity measurements, but optimized sampling strategies and point-spread function engineering can facilitate rapid volume imaging of neural activity within this constraint. We also discuss new computational strategies for processing and analyzing volume imaging data of increasing size and complexity. Together, optical and computational advances are providing a broader view of neural circuit dynamics and helping elucidate how brain regions work in concert to support behavior.

Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain.

Two-photon calcium imaging provides an optical readout of neuronal activity in populations of neurons with subcellular resolution. However, conventional two-photon imaging systems are limited in their field of view to ∼1 mm(2), precluding the visualization of multiple cortical areas simultaneously. Here, we demonstrate a two-photon microscope with an expanded field of view (>9.5 mm(2)) for rapidly reconfigurable simultaneous scanning of widely separated populations of neurons. We custom designed and assembled an optimized scan engine, objective, and two independently positionable, temporally multiplexed excitation pathways. We used this new microscope to measure activity correlations between two cortical visual areas in mice during visual processing.

Improving data quality in neuronal population recordings.

Understanding how the brain operates requires understanding how large sets of neurons function together. Modern recording technology makes it possible to simultaneously record the activity of hundreds of neurons, and technological developments will soon allow recording of thousands or tens of thousands. As with all experimental techniques, these methods are subject to confounds that complicate the interpretation of such recordings, and could lead to erroneous scientific conclusions. Here we discuss methods for assessing and improving the quality of data from these techniques and outline likely future directions in this field.

Persistent changes in spontaneous firing of Purkinje neurons triggered by the nitric oxide signaling cascade.

Many types of neurons fire spontaneously because of the activity of pacemaking ion channels. Although endogenous firing can serve as a persistent signal to downstream targets, little attention has been paid to factors that might modulate such intrinsic electrical activity. We tested for modulation of spontaneous firing of Purkinje neurons in cerebellar slices under conditions in which principal synaptic inputs were blocked. Loose-patch recordings from single neurons show that sustained (>40 min) increases in the spontaneous firing rate can be triggered by activation of the nitric oxide-cGMP signaling pathway. Inhibitors of soluble guanylate cyclase and protein kinase G block this modulation. Increases in firing rate are also observed after stimulation of parallel fibers but not in response to basket cell activity. These findings elucidate a novel role for the nitric oxide-cGMP signaling cascade in the brain. This mechanism could permit long-term adjustments in the baseline firing rate of endogenously active neurons in response to changes in afferent activity.

An ultra small array of electrodes for stimulating multiple inputs into a single neuron.

We have developed an ultra small, translucent array of electrodes for use in the parasaggital cerebellar slice preparation. This positionable array is capable of stimulating multiple independent bundles of parallel fibers (PFs), which synapse onto a single Purkinje neuron. On a silicon substrate, a low-stress silicon nitride film was used both as a structural layer and as electrical insulation. Evaporated gold pads and interconnects were sandwiched between two such layers. A bulk anisotropic silicon etch released the individual arrays. The electrodes are supported within a 2-microm-thick cantilever of translucent silicon nitride. In one design, eight 4-microm-wide square electrodes are arranged on 8-microm-centers. Another design, half the scale of the first, was also tested. The array was mounted on a micromanipulator and can be visualized by an upright microscope. It can then be positioned in the dendritic arbor of a Purkinje neuron while not disturbing a recording pipette at the soma. Paired-pulse facilitation experiments have confirmed that the electrodes are capable of stimulating non-overlapping bundles of PFs. This device will be useful for exploring spatiotemporal synaptic integration in single neurons. Potential applications in experiments on cerebellar LTD are also discussed.

Synapse-specific control of experience-dependent plasticity by presynaptic NMDA receptors.

Sensory experience orchestrates the development of cortical circuitry by adaptively modifying neurotransmission and synaptic connectivity. However, the mechanisms underlying these experience-dependent modifications remain elusive. Here we demonstrate that visual experience suppresses a presynaptic NMDA receptor (preNMDAR)-mediated form of timing-dependent long-term depression (tLTD) at visual cortex layer (L) 4-2/3 synapses. This tLTD can be maintained during development, or reinstated in adulthood, by sensory deprivation. The changes in tLTD are mirrored by changes in glutamate release; visual deprivation enhances both tLTD and glutamate release. These effects require the GluN3A NMDAR subunit, the levels of which are increased by visual deprivation. Further, by coupling the pathway-specific optogenetic induction of tLTD with cell-type-specific NMDAR deletion, we find that visual experience modifies preNMDAR-mediated plasticity specifically at L4-L2/3 synapses.