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How have animals managed to maintain metabolically expensive brains given the volatile and fleeting availability of calories in the natural world? Here we review studies in support of three strategies that involve: 1) a reallocation of energy from peripheral tissues and functions to cover the costs of the brain, 2) an implementation of energy-efficient neural coding, enabling the brain to operate at reduced energy costs, and 3) efficient use of costly neural resources during food scarcity. Collectively, these studies reveal a heterogeneous set of energy-saving mechanisms that make energy-costly brains fit for survival.

In this issue of Neuron, Jordan and Keller (2020) explore subthreshold computations underlying predictive coding using whole-cell recordings in mouse visual cortex. Their findings suggest that layer 2/3, but not layer 5/6, neurons compute prediction errors by subtracting predicted and actual visual flow inputs generated by locomotion.

In mammals with binocular vision, integration of the left and right visual scene relies on information in the center visual field, which are relayed from each retina in parallel and merge in the primary visual cortex (V1) through the convergence of ipsi- and contralateral geniculocortical inputs as well as transcallosal projections between two visual cortices. The developmental assembly of this binocular circuit, especially the transcal-losal pathway, remains incompletely understood. Using genetic methods in mice, we found that several days before eye-opening, retinal and callosal activities drive massive apoptosis of GABAergic chandelier cells (ChCs) in the binocular region of V1. Blockade of ChC elimination resulted in a contralateral eye-domi-nated V1 and deficient binocular vision. As pre-vision retinal activities convey the left-right organization of the visual field, their regulation of ChC density through the transcallosal pathway may prime a nascent binocular territory for subsequent experience-driven tuning during the post-vision critical period.

During navigation, animals integrate sensory information with body movements to guide actions. The impact of both navigational and movement-related signals on cortical visual information processing remains largely unknown. We review recent studies in awake rodents that have revealed navigation-related signals in the primary visual cortex (V1) including speed, distance travelled and head-orienting movements. Both cortical and subcortical inputs convey self-motion related information to V1 neurons: for example, top-down inputs from secondary motor and retrosplenial cortices convey information about head movements and spatial expectations. Within V1, subtypes of inhibitory neurons are critical for the integration of navigation-related and visual signals. We conclude with potential functional roles of navigation-related signals in V1 including gain control, motor error signals and predictive coding.

SummaryTo initiate goal-directed behavior, animals must transform sensory cues into motor commands that generate appropriately timed actions. Sensorimotor transformations along the cerebellar-thalamocortical pathway are thought to shape motor cortical output and movement timing, but whether this pathway initiates goal-directed movement remains poorly understood. Here, we recorded and perturbed activity in cerebellar-recipient regions of motor thalamus (dentate / interpositus nucleus-recipient regions, MThDN/IPN) and primary motor cortex (M1) in mice trained to execute a cued forelimb lever push task for reward. MThDN/IPN population responses were dominated by a time-locked increase in activity immediately prior to movement that was temporally uncoupled from cue presentation, providing a fixed latency feedforward motor timing signal to M1FL. Blocking MThDN/IPN output suppressed cued movement initiation. Stimulating the MThDN/IPN thalamocortical pathway in the absence of the cue recapitulated cue-evoked M1 membrane potential dynamics and forelimb behavior in the learned behavioral context, but generated semi-random movements in an altered behavioral context. Thus, cerebellar-recipient motor thalamocortical input to M1 is indispensable for the generation of motor commands that initiate goal-directed movement, refining our understanding of how the cerebellar-thalamocortical pathway contributes to movement timing.

Executing learned motor behaviors often requires the transformation of sensory cues into patterns of motor commands that generate appropriately timed actions. The cerebellum and thalamus are two key areas involved in shaping cortical output and movement, but the contribution of a cerebellar-thalamocortical pathway to voluntary movement initiation remains poorly understood. Here, we investigated how an auditory "go cue" transforms thalamocortical activity patterns and how these changes relate to movement initiation. Population responses in dentate/interpositus-recipient regions of motor thalamus reflect a time-locked increase in activity immediately prior to movement initiation that is temporally uncoupled from the go cue, indicative of a fixed-latency feedforward motor timing signal. Blocking cerebellar or motor thalamic output suppresses movement initiation, while stimulation triggers movements in a behavioral context-dependent manner. Our findings show how cerebellar output, via the thalamus, shapes cortical activity patterns necessary for learned context-dependent movement initiation.

Neuroscience research related to functionality, connectivity and metabolism of neuronal circuits, individual neuronal cells and sub-cellular structures, nowadays, experiences a burgeoning need to develop techniques for the detailed investigation inside the complexity of the living matter. Particularly, high-resolution observations combined with an extended depth of penetration in tissue represents an ongoing challenge. Holographic control of light propagation in complex media opens a promising way to overcome this technological barrier via exploiting multimode fibres as hair-thin, minimally-invasive endoscopes. This concept allows for more than one order of magnitude reduction of the instrument’s footprint and a significant enhancement of imaging resolution, compared with current minimally invasive endoscopes. Here, we demonstrate a compact and high-speed system for fluorescent imaging at the tip of a fibre. The instrument’s performance reaches micron-scale resolution across a field of view 50 micrometres, yielding 7-kilopixel image information at a rate of 3.5 frames per second. The resolution limit is dictated only by the numerical aperture of the fibre probe, and the contrast/pureness of the focal points, utilised for raster-scanning regime, approach the theoretical limits for phase-only holographic wavefront shaping. The achieved performance allowed for in-vivo observations of neuronal somata and processes, residing deep inside the visual cortex and hippocampus of an animal model with minimal damage to the tissue surrounding the fibre penetration area. We believe that this demonstration represents an important step towards implementations of various advanced forms of imaging through multimode fibre based endoscopes to address numerous key challenges in neuroscience.

Progress in neuroscience constantly relies on the development of new techniques to investigate the complex dynamics of neuronal networks. An ongoing challenge is to achieve minimally-invasive and high-resolution observations of neuronal activity in vivo inside deep brain areas. A perspective strategy is to utilise holographic control of light propagation in complex media, which allows converting a hair-thin multimode optical fibre into an ultra-narrow imaging tool. Compared to current endoscopes based on GRIN lenses or fibre bundles, this concept offers a footprint reduction exceeding an order of magnitude, together with a significant enhancement in resolution. We designed a compact and high-speed system for fluorescent imaging at the tip of a fibre, achieving micron-scale resolution across a 50 um field of view, and yielding 7-kilopixel images at a rate of 3.5 frames/s. Furthermore, we demonstrate in vivo observations of cell bodies and processes of inhibitory neurons within deep layers of the visual cortex and hippocampus of anesthetised mice. This study forms the basis for several perspective techniques of modern microscopy to be delivered deep inside the tissue of living animal models while causing minimal impact on its structural and functional properties., Comment: 10 pages, 2 figures, Supplementary movie: https://drive.google.com/file/d/1Fm0G3TAIC49LVX6FaEiAtlefkWx1T2a5/view

Neural circuit assembly relies on the precise synchronization of developmental processes, such as cell migration and axon targeting, but the cell-autonomous mechanisms coordinating these events remain largely unknown. Here we found that different classes of interneurons use distinct routes of migration to reach the embryonic cerebral cortex. Somatostatin-expressing interneurons that migrate through the marginal zone develop into Martinotti cells, one of the most distinctive classes of cortical interneurons. For these cells, migration through the marginal zone is linked to the development of their characteristic layer 1 axonal arborization. Altering the normal migratory route of Martinotti cells by conditional deletion of Mafb—a gene that is preferentially expressed by these cells—cell-autonomously disrupts axonal development and impairs the function of these cells in vivo. Our results suggest that migration and axon targeting programs are coupled to optimize the assembly of inhibitory circuits in the cerebral cortex., This work was supported by a grant from the European Research Council (ERC-2011-AdG 293683) to O.M., Marie Curie Actions of the European Union’s FP7 program (MC-CIG 631770) to N.R. and (IEF 624461) J.P., and funds from the Shirley Foundation, Patrick Wild Center, RS MacDonald Charitable Trust, and Simons Initiative for the Developing Brain to N.R. L.L. was the recipient of an EMBO long-term postdoctoral fellowship. N.R. is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (102857/Z/13/Z).

Our ability to learn relies on the potential of neuronal circuits to change through experience. Recent advances in genetic tools and in vivo imaging have enabled novel investigations into the mechanisms of plasticity at the level of neuronal circuits. This chapter describes an experimental method for long-term activity monitoring of large neuronal populations, activity, with single-cell resolution, in the cortex of awake behaving mice. By combining genetically encoded calcium indicators with two-photon imaging, this technique allows researchers to relate the activity of subtypes of neurons directly to the animal's behavior over temporal scales from hundreds of milliseconds to several weeks. We detail the key experimental aspects of long-term, repeated two-photon calcium imaging: surgical procedures for chronic craniotomies, repeated image acquisition, and critical analysis parameters for two-photon calcium imaging data. Finally, we review recent applications of this technique to study the plasticity of neuronal circuits in the primary visual cortex.

In vivo calcium imaging has become a method of choice to image neuronal population activity throughout the nervous system. These experiments generate large sequences of images. Their analysis is computationally intensive and typically involves motion correction, image segmentation into regions of interest (ROIs), and extraction of fluorescence traces from each ROI. Out of focus fluorescence from surrounding neuropil and other cells can strongly contaminate the signal assigned to a given ROI. In this study, we introduce the FISSA toolbox (Fast Image Signal Separation Analysis) for neuropil decontamination. Given pre-defined ROIs, the FISSA toolbox automatically extracts the surrounding local neuropil and performs blind-source separation with non-negative matrix factorization. Using both simulated and in vivo data, we show that this toolbox performs similarly or better than existing published methods. FISSA requires only little RAM, and allows for fast processing of large datasets even on a standard laptop. The FISSA toolbox is available in Python, with an option for MATLAB format outputs, and can easily be integrated into existing workflows. It is available from Github and the standard Python repositories.

Cortical responses to sensory stimuli are modulated by behavioral state. In the primary visual cortex (V1), visual responses of pyramidal neurons increase during locomotion. This response gain was suggested to be mediated through inhibitory neurons, resulting in the disinhibition of pyramidal neurons. Using in vivo two-photon calcium imaging in layers 2/3 and 4 in mouse V1, we reveal that locomotion increases the activity of vasoactive intestinal peptide (VIP), somatostatin (SST) and parvalbumin (PV)-positive interneurons during visual stimulation, challenging the disinhibition model. In darkness, while most VIP and PV neurons remained locomotion responsive, SST and excitatory neurons were largely non-responsive. Context-dependent locomotion responses were found in each cell type, with the highest proportion among SST neurons. These findings establish that modulation of neuronal activity by locomotion is context-dependent and contest the generality of a disinhibitory circuit for gain control of sensory responses by behavioral state.

Cortical responses to sensory stimuli are modulated by behavioral state. In the primary visual cortex (V1), visual responses of pyramidal neurons increase during locomotion. This response gain was suggested to be mediated through inhibitory neurons, resulting in the disinhibition of pyramidal neurons. Using in vivo two-photon calcium imaging in layers 2/3 and 4 in mouse V1, we reveal that locomotion increases the activity of vasoactive intestinal peptide (VIP), somatostatin (SST) and parvalbumin (PV)-positive interneurons during visual stimulation, challenging the disinhibition model. In darkness, while most VIP and PV neurons remained locomotion responsive, SST and excitatory neurons were largely non-responsive. Context-dependent locomotion responses were found in each cell type, with the highest proportion among SST neurons. These findings establish that modulation of neuronal activity by locomotion is context-dependent and contest the generality of a disinhibitory circuit for gain control of sensory responses by behavioral state. DOI: http://dx.doi.org/10.7554/eLife.14985.001, eLife digest How we perceive what we see depends on the context in which we see it, such as what we are doing at the time. For example, we perceive a park landscape differently when we are running through it than when we are sitting on a park bench. Behavior can also alter neuronal responses in the brain. Indeed, the neurons in the part of the brain that receives information related to vision (known as the visual cortex) respond differently to visual stimuli when an animal is moving compared to when the animal is still. However, while some recent studies revealed that specific types of neurons become more or less responsive during movement, others reported the opposite results. One hypothesis that would explain these contradictory findings would be if the way that behavior, in this case movement, affects neuronal responses also depends on the external context in which the movement happens. Now, Pakan et al. have tested this hypothesis by imaging the activity of different types of neurons in the primary visual cortex of mice that were either running on a treadmill or staying still. The experiments were conducted in two different contexts: in total darkness (in which the mice could not see) and in the presence of display screens (which provided the mice with visual stimulation). Pakan et al. confirmed that running does indeed affect the activity of specific neurons in different ways in different contexts. For example, when the mice received visual stimulation, the three main classes of neurons that send inhibitory signals in the visual cortex became more active during running. However, when the mouse ran in the dark, two of these neuron types became more active during running while the third type of neuron was unresponsive. This finding reveals more about the dynamic nature of inhibitory activity that strongly depends on the animal’s behaviour. It also shows how these neurons influence the excitatory neurons in the visual cortex, which send information to the rest of the brain for further processing towards perception. The next step will be to identify what precise mechanism makes these neurons respond differently in unique contexts, and to tease apart how these movement-dependent signals affect the way animals perceive visual stimuli. DOI: http://dx.doi.org/10.7554/eLife.14985.002

Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.

The individual functional properties and spatial arrangement of afferent synaptic inputs on dendrites have a critical role in the processing of information by neurons in the mammalian brain. Although recent work has identified visually-evoked local dendritic calcium signals in the rodent visual cortex, sensory-evoked signalling on the level of dendritic spines, corresponding to individual afferent excitatory synapses, remains unexplored. Here we used a new variant of high-resolution two-photon imaging to detect sensory-evoked calcium transients in single dendritic spines of mouse cortical neurons in vivo. Calcium signals evoked by sound stimulation required the activation of NMDA (N-methyl-D-aspartate) receptors. Active spines are widely distributed on basal and apical dendrites and pure-tone stimulation at different frequencies revealed both narrowly and widely tuned spines. Notably, spines tuned for different frequencies were highly interspersed on the same dendrites: even neighbouring spines were mostly tuned to different frequencies. Thus, our results demonstrate that NMDA-receptor-dependent single-spine synaptic inputs to the same dendrite are highly heterogeneous. Furthermore, our study opens the way for in vivo mapping of functionally defined afferent sensory inputs with single-synapse resolution.

Eye-opening represents a turning point in the function of the visual cortex. Before eye-opening, the visual cortex is largely devoid of sensory inputs and neuronal activities are generated intrinsically. After eye-opening, the cortex starts to integrate visual information. Here we used in vivo two-photon calcium imaging to explore the developmental changes of the mouse visual cortex by analyzing the ongoing spontaneous activity. We found that before eye-opening, the activity of layer 2/3 neurons consists predominantly of slow wave oscillations. These waves were first detected at postnatal day 8 (P8). Their initial very low frequency (0.01 Hz) gradually increased during development to ≈0.5 Hz in adults. Before eye-opening, a large fraction of neurons (>75%) was active during each wave. One day after eye-opening, this dense mode of recruitment changed to a sparse mode with only 36% of active neurons per wave. This was followed by a progressive decrease during the following weeks, reaching 12% of active neurons per wave in adults. The possible role of visual experience for this process of sparsification was investigated by analyzing dark-reared mice. We found that sparsification also occurred in these mice, but that the switch from a dense to a sparse activity pattern was delayed by 3–4 days as compared with normally-reared mice. These results reveal a modulatory contribution of visual experience during the first days after eye-opening, but an overall dominating role of intrinsic factors. We propose that the transformation in network activity from dense to sparse is a prerequisite for the changed cortical function at eye-opening.

The functional specificity of callosal connections was investigated in visual areas 17 and 18 of adult cats, by combining in vivo optical imaging of intrinsic signals with labeling of callosal axons. Local injections of neuronal tracers were performed in one hemisphere and eight single callosal axons were reconstructed in the opposite hemisphere. The distributions of injection sites and callosal axon terminals were analyzed with respect to functional maps in both hemispheres. Typically, each callosal axon displayed 2 or 3 clusters of synaptic boutons in layer II/III and the upper part of layer IV. These clusters were preferentially distributed in regions representing the same orientation and the same visuotopic location as that at the corresponding injection sites in the opposite hemisphere. The spatial distribution of these clusters was elongated and its main axis correlated well with the preferred orientation at the injection site. These results demonstrate a specific organization of interhemispheric axons that link cortical regions representing the same orientation and the same location of visual stimuli. Visual callosal connections are thus likely involved in the processing of coherent information in terms of shape and position along the midline of the visual field, which may facilitate the fusion of both hemifields into the percept of a single visual scene.

Calcium imaging has revolutionized the approaches for functional analyses in the living brain of animal experimental models. Changes in intracellular calcium concentration are strictly linked to the electrical activity in neurons and produce signals that are effectively detected by optical methods. Distinctive features of fluorescence-based calcium imaging are its high temporal resolution in the millisecond range and its high spatial resolution in the micrometer range. Recent progress includes the development of fluorometric calcium sensors, new approaches for targeted labeling with these sensors and the implementation of powerful imaging techniques, especially two-photon microscopy. An important and rapidly evolving field of current research is the use of calcium imaging for the analysis of in vivo mouse models for various brain diseases, such as Alzheimer's disease, stroke and epilepsy.

The contribution of interhemispheric connections to functional maps in cat visual cortex was investigated by using optical imaging of intrinsic signals. In order to isolate the functional inputs arriving via the corpus callosum (CC) from other inputs, we used the split-chiasm preparation. The regions activated through the CC in visual areas 17 (A17) and 18 (A18) were localized and characterized by stimulating monocularly split-chiasm cats with moving, high contrast oriented gratings. We found that the CC mediates the activation of orientation selective domains in the transition zone (TZ) between A17 and A18 and occasionally within portions of both of these areas. We observed transcallosally activated orientation domains all along the TZ without any obvious interruption, and these domains were arranged around “pinwheel” centers. Interestingly, the TZ was divided in two parallel regions, which resemble A17 and A18 in their preferred temporal and spatial frequencies. Finally, we demonstrated that orientation maps evoked through the transcallosal and geniculo-cortical pathways were similar within the TZ, indicating a convergence of inputs of matching orientations in this region. These results contribute to a better understanding of the role of the CC in visual perception of orientations and shapes, at the level of the visual cortex.

In cat visual cortex, neurons acquire progressively mature functional properties during the first postnatal months. The aim of this study was to analyze the development of astrocytes during this period. The patterns of expression of the glial fibrillary acidic protein (GFAP) as well as of two gap junction proteins expressed in astrocytes, connexin43 (Cx43) and connexin30 (Cx30), were investigated by immunohistochemistry and optical density measurements, in visual cortical areas 17 and 18 at four different ages: 2 weeks (postnatal days 12 to 15, P12-15), 1 month (P27-31), 2 months (P60-62) and beyond 1 year. Since visual experience is a key factor for neural development, the patterns of expression of these three proteins were studied both in normally-reared and monocularly deprived animals. Interestingly, the distribution of GFAP, Cx43 and Cx30 was found to change dramatically but independently of visual experience, during postnatal development, even beyond P60. During the first postnatal month, GFAP and Cx43 were mainly localized in the white matter underlying the visual cortical areas 17 and 18. Then, their distributions evolved similarly with a progressive decrease of their density in the white matter associated with an increase in the cortex. Connexin30 expression appeared only from the second postnatal month, strictly in the cortex and with a laminar distribution which was similar to that of Cx43 at the same age. In adults, a specific laminar distribution was observed, that was identical for GFAP, Cx43 and Cx30: their density was higher in layers II/III and V than in the other cortical layers.

In the adult cat, axons running through the corpus callosum interconnect the border between the visual cortical areas 17 and 18 (A17 and A18) of both hemispheres. This specific pattern emerges during postnatal development, under normal viewing conditions (NR), from the elimination of initially exuberant callosal projections. In contrast, if the postnatal visual experience is monocular from birth (MD), juvenile callosal projections are stabilised throughout A17 and A18. The present study aimed at using such a model in vivo to find indications of a contribution of glial cells in the shaping of projections in the developing CNS through interactions with neurones, both in normal and pathological conditions. As a first stage, the distribution and the morphology of microglial cells and astrocytes were investigated from 2 weeks to adulthood. Microglial cells, stained with isolectin-B4, were clustered in the white matter below A17 and A18. Until one month, these clustered cells displayed an ameboid morphology in NR group, while they were more ramified in MD animals. Their phenotype thus depends on the postnatal visual experience, which indicates that microglial cells may interact with axons of visual neurones. It also suggests that they may differentially contribute to the elimination and the stabilisation of juvenile exuberant callosal fibres in NR and MD animals respectively. Beyond one month, microglial cells were very ramified in both experimental groups. Astrocytes were labelled with a GFAP-antibody. The distributions of connexins 43 (Cx43) and 30 (Cx30), the main proteic components of gap junction channels in astrocytes, were also investigated using specific antibodies. Both in NR and MD groups, until 1 month, GFAP-positive astrocytes and Cx43 were mainly localised within the subcortical white matter. Then GFAP, Cx43 and Cx30 stainings progressively appeared within the cortex, throughout A17 and A18 but with a differential laminar expression according to the age. Thus, the distributions of both astrocytes and connexins changed with age; however, the monocular occlusion had no visible effect. This suggests that astrocytes may contribute to the postnatal development of neuronal projections to the primary visual cortex, including visual callosal projections.

Dendritic spines arise as small protrusions from the dendritic shaft of various types of neuron and receive inputs from excitatory axons. Ever since dendritic spines were first described in the nineteenth century, questions about their function have spawned many hypotheses. In this review, we introduce understanding of the structural and biochemical properties of dendritic spines with emphasis on components studied with imaging methods. We then explore advances in in vivo imaging methods that are allowing spine activity to be studied in living tissue, from super-resolution techniques to calcium imaging. Finally, we review studies on spine structure and function in vivo. These new results shed light on the development, integration properties and plasticity of spines.

The accumulation of amyloid-β in the brain is an essential feature of Alzheimer's disease. However, the impact of amyloid-β-accumulation on neuronal dysfunction on the single cell level in vivo is poorly understood. Here we investigate the progression of amyloid-β load in relation to neuronal dysfunction in the visual system of the APP23×PS45 mouse model of Alzheimer's disease. Using in vivo two-photon calcium imaging in the visual cortex, we demonstrate that a progressive deterioration of neuronal tuning for the orientation of visual stimuli occurs in parallel with the age-dependent increase of the amyloid-β load. Importantly, we find this deterioration only in neurons that are hyperactive during spontaneous activity. This impairment of visual cortical circuit function also correlates with pronounced deficits in visual-pattern discrimination. Together, our results identify distinct stages of decline in sensory cortical performance in vivo as a function of the increased amyloid-β-load., The amyloid-β-peptide is pivotal to the pathology of Alzheimer's disease, but its mechanism of action remains uncertain. This study utilizes in vivo two-photon calcium imaging to investigate the effects of this peptide on single cortical neurons of the visual cortex in a mouse model of Alzheimer's disease.

Neurons in cortical sensory regions receive modality-specific information through synapses that are located on their dendrites. Recently, the use of two-photon microscopy combined with whole-cell recordings has helped to identify visually evoked dendritic calcium signals in mouse visual cortical neurons in vivo. The calcium signals are restricted to small dendritic domains ('hotspots') and they represent visual synaptic inputs that are highly tuned for orientation and direction. This protocol describes the experimental procedures for the recording and the analysis of these visually evoked dendritic calcium signals. The key points of this method include delivery of fluorescent calcium indicators through the recording patch pipette, selection of an appropriate optical plane with many dendrites, hyperpolarization of the membrane potential and two-photon imaging. The whole protocol can be completed in 5-6 h, including 1-2 h of two-photon calcium imaging in combination with stable whole-cell recordings.

In sensory cortex regions, neurons are tuned to specific stimulus features. For example, in the visual cortex, many neurons fire predominantly in response to moving objects of a preferred orientation. However, the characteristics of the synaptic input that cortical neurons receive to generate their output firing pattern remain unclear. Here we report a novel approach for the visualization and functional mapping of sensory inputs to the dendrites of cortical neurons in vivo. By combining high-speed two-photon imaging with electrophysiological recordings, we identify local subthreshold calcium signals that correspond to orientation-specific synaptic inputs. We find that even inputs that share the same orientation preference are widely distributed throughout the dendritic tree. At the same time, inputs of different orientation preference are interspersed, so that adjacent dendritic segments are tuned to distinct orientations. Thus, orientation-tuned neurons can compute their characteristic firing pattern by integrating spatially distributed synaptic inputs coding for multiple stimulus orientations.

A decade after the introduction of genetically encoded Ca2+ indicator proteins (GECIs), a new generation of improved GECIs demonstrates their usefulness for the functional analysis of the mammalian brain in vivo.

Previous studies of the ferret visual cortex indicate that the development of direction selectivity requires visual experience. Here, we used two-photon calcium imaging to study the development of direction selectivity in layer 2/3 neurons of the mouse visual cortex in vivo. Surprisingly, just after eye opening nearly all orientation-selective neurons were also direction selective. During later development, the number of neurons responding to drifting gratings increased in parallel with the fraction of neurons that were orientation, but not direction, selective. Our experiments demonstrate that direction selectivity develops normally in dark-reared mice, indicating that the early development of direction selectivity is independent of visual experience. Furthermore, remarkable functional similarities exist between the development of direction selectivity in cortical neurons and the previously reported development of direction selectivity in the mouse retina. Together, these findings provide strong evidence that the development of orientation and direction selectivity in the mouse brain is distinctly different from that in ferrets.

SUMMARY In the mammalian brain, calcium signals in dendritic spines are involved in many neuronal functions, particularly in the induction of synaptic plasticity. Recent studies have identified sensory stimulationevoked spine calcium signals in cortical neurons in vivo. However, spine signaling during ongoing cortical activity in the absence of sensory input, which is essential for important functions like memory consolidation, is not well understood. Here, by using in vivo two-photon imaging of auditory cortical neurons, we demonstrate that subthreshold, NMDAreceptor-dependent spine calcium signals are abundant during up states, but almost absent during down states. In each neuron, about 500 nonclustered spines, which are widely dispersed throughout the dendritic field, are on average active during an up state. The same subset of spines is reliably active during both sensory stimulation and up states. Thus, spontaneously recurring up states evoke in these spines ‘‘patterned’’ calcium activity that may control consolidation of synaptic strength following epochs of sensory stimulation.