Neuronal plasticity: beyond the critical period.

Neuronal plasticity in the brain is greatly enhanced during critical periods early in life and was long thought to be rather limited thereafter. Studies in primary sensory areas of the neocortex have revealed a substantial degree of plasticity in the mature brain, too. Often, plasticity in the adult neocortex lies dormant but can be reactivated by modifications of sensory input or sensory-motor interactions, which alter the level and pattern of activity in cortical circuits. Such interventions, potentially in combination with drugs targeting molecular brakes on plasticity present in the adult brain, might help recovery of function in the injured or diseased brain.

Mouse prefrontal cortex represents learned rules for categorization.

The ability to categorize sensory stimuli is crucial for an animal's survival in a complex environment. Memorizing categories instead of individual exemplars enables greater behavioural flexibility and is computationally advantageous. Neurons that show category selectivity have been found in several areas of the mammalian neocortex1-4, but the prefrontal cortex seems to have a prominent role4,5 in this context. Specifically, in primates that are extensively trained on a categorization task, neurons in the prefrontal cortex rapidly and flexibly represent learned categories6,7. However, how these representations first emerge in naive animals remains unexplored, leaving it unclear whether flexible representations are gradually built up as part of semantic memory or assigned more or less instantly during task execution8,9. Here we investigate the formation of a neuronal category representation throughout the entire learning process by repeatedly imaging individual cells in the mouse medial prefrontal cortex. We show that mice readily learn rule-based categorization and generalize to novel stimuli. Over the course of learning, neurons in the prefrontal cortex display distinct dynamics in acquiring category selectivity and are differentially engaged during a later switch in rules. A subset of neurons selectively and uniquely respond to categories and reflect generalization behaviour. Thus, a category representation in the mouse prefrontal cortex is gradually acquired during learning rather than recruited ad hoc. This gradual process suggests that neurons in the medial prefrontal cortex are part of a specific semantic memory for visual categories.

DeepD3, an open framework for automated quantification of dendritic spines.

Dendritic spines are the seat of most excitatory synapses in the brain, and a cellular structure considered central to learning, memory, and activity-dependent plasticity. The quantification of dendritic spines from light microscopy data is usually performed by humans in a painstaking and error-prone process. We found that human-to-human variability is substantial (inter-rater reliability 82.2±6.4%), raising concerns about the reproducibility of experiments and the validity of using human-annotated 'ground truth' as an evaluation method for computational approaches of spine identification. To address this, we present DeepD3, an open deep learning-based framework to robustly quantify dendritic spines in microscopy data in a fully automated fashion. DeepD3's neural networks have been trained on data from different sources and experimental conditions, annotated and segmented by multiple experts and they offer precise quantification of dendrites and dendritic spines. Importantly, these networks were validated in a number of datasets on varying acquisition modalities, species, anatomical locations and fluorescent indicators. The entire DeepD3 open framework, including the fully segmented training data, a benchmark that multiple experts have annotated, and the DeepD3 model zoo is fully available, addressing the lack of openly available datasets of dendritic spines while offering a ready-to-use, flexible, transparent, and reproducible spine quantification method.

Functional network topography of the medial entorhinal cortex.

The medial entorhinal cortex (MEC) creates a map of local space, based on the firing patterns of grid, head-direction (HD), border, and object-vector (OV) cells. How these cell types are organized anatomically is debated. In-depth analysis of this question requires collection of precise anatomical and activity data across large populations of neurons during unrestrained behavior, which neither electrophysiological nor previous imaging methods fully afford. Here, we examined the topographic arrangement of spatially modulated neurons in the superficial layers of MEC and adjacent parasubiculum using miniaturized, portable two-photon microscopes, which allow mice to roam freely in open fields. Grid cells exhibited low levels of co-occurrence with OV cells and clustered anatomically, while border, HD, and OV cells tended to intermingle. These data suggest that grid cell networks might be largely distinct from those of border, HD, and OV cells and that grid cells exhibit strong coupling among themselves but weaker links to other cell types.

Functional and structural features of L2/3 pyramidal cells continuously covary with pial depth in mouse visual cortex.

Pyramidal cells of neocortical layer 2/3 (L2/3 PyrCs) integrate signals from numerous brain areas and project throughout the neocortex. These PyrCs show pial depth-dependent functional and structural specializations, indicating participation in different functional microcircuits. However, whether these depth-dependent differences result from separable PyrC subtypes or whether their features display a continuum correlated with pial depth is unknown. Here, we assessed the stimulus selectivity, electrophysiological properties, dendritic morphology, and excitatory and inhibitory connectivity across the depth of L2/3 in the binocular visual cortex of mice. We find that the apical, but not the basal dendritic tree structure, varies with pial depth, which is accompanied by variation in subthreshold electrophysiological properties. Lower L2/3 PyrCs receive increased input from L4, while upper L2/3 PyrCs receive a larger proportion of intralaminar input. In vivo calcium imaging revealed a systematic change in visual responsiveness, with deeper PyrCs showing more robust responses than superficial PyrCs. Furthermore, deeper PyrCs are more driven by contralateral than ipsilateral eye stimulation. Importantly, the property value transitions are gradual, and L2/3 PyrCs do not display discrete subtypes based on these parameters. Therefore, L2/3 PyrCs' multiple functional and structural properties systematically correlate with their depth, forming a continuum rather than discrete subtypes.

Transplanted embryonic neurons integrate into adult neocortical circuits.

The ability of the adult mammalian brain to compensate for neuronal loss caused by injury or disease is very limited. Transplantation aims to replace lost neurons, but the extent to which new neurons can integrate into existing circuits is unknown. Here, using chronic in vivo two-photon imaging, we show that embryonic neurons transplanted into the visual cortex of adult mice mature into bona fide pyramidal cells with selective pruning of basal dendrites, achieving adult-like densities of dendritic spines and axonal boutons within 4-8 weeks. Monosynaptic tracing experiments reveal that grafted neurons receive area-specific, afferent inputs matching those of pyramidal neurons in the normal visual cortex, including topographically organized geniculo-cortical connections. Furthermore, stimulus-selective responses refine over the course of many weeks and finally become indistinguishable from those of host neurons. Thus, grafted neurons can integrate with great specificity into neocortical circuits that normally never incorporate new neurons in the adult brain.

Disparity Sensitivity and Binocular Integration in Mouse Visual Cortex Areas.

Binocular disparity, the difference between the two eyes' images, is a powerful cue to generate the 3D depth percept known as stereopsis. In primates, binocular disparity is processed in multiple areas of the visual cortex, with distinct contributions of higher areas to specific aspects of depth perception. Mice, too, can perceive stereoscopic depth, and neurons in primary visual cortex (V1) and higher-order, lateromedial (LM) and rostrolateral (RL) areas were found to be sensitive to binocular disparity. A detailed characterization of disparity tuning across mouse visual areas is lacking, however, and acquiring such data might help clarifying the role of higher areas for disparity processing and establishing putative functional correspondences to primate areas. We used two-photon calcium imaging in female mice to characterize the disparity tuning properties of neurons in visual areas V1, LM, and RL in response to dichoptically presented binocular gratings, as well as random dot correlograms (RDC). In all three areas, many neurons were tuned to disparity, showing strong response facilitation or suppression at optimal or null disparity, respectively, even in neurons classified as monocular by conventional ocular dominance (OD) measurements. Neurons in higher areas exhibited broader and more asymmetric disparity tuning curves compared with V1, as observed in primate visual cortex. Finally, we probed neurons' sensitivity to true stereo correspondence by comparing responses to correlated RDC (cRDC) and anticorrelated RDC (aRDC). Area LM, akin to primate ventral visual stream areas, showed higher selectivity for correlated stimuli and reduced anticorrelated responses, indicating higher-level disparity processing in LM compared with V1 and RL.SIGNIFICANCE STATEMENT A major cue for inferring 3D depth is disparity between the two eyes' images. Investigating how binocular disparity is processed in the mouse visual system will not only help delineating the role of mouse higher areas for visual processing, but also shed light on how the mammalian brain computes stereopsis. We found that binocular integration is a prominent feature of mouse visual cortex, as many neurons are selectively and strongly modulated by binocular disparity. Comparison of responses to correlated and anticorrelated random dot correlograms (RDC) revealed that lateromedial area (LM) is more selective to correlated stimuli, while less sensitive to anticorrelated stimuli compared with primary visual cortex (V1) and rostrolateral area (RL), suggesting higher-level disparity processing in LM, resembling primate ventral visual stream areas.

Grid cells and cortical representation.

One of the grand challenges in neuroscience is to comprehend neural computation in the association cortices, the parts of the cortex that have shown the largest expansion and differentiation during mammalian evolution and that are thought to contribute profoundly to the emergence of advanced cognition in humans. In this Review, we use grid cells in the medial entorhinal cortex as a gateway to understand network computation at a stage of cortical processing in which firing patterns are shaped not primarily by incoming sensory signals but to a large extent by the intrinsic properties of the local circuit.

What is memory? The present state of the engram.

The mechanism of memory remains one of the great unsolved problems of biology. Grappling with the question more than a hundred years ago, the German zoologist Richard Semon formulated the concept of the engram, lasting connections in the brain that result from simultaneous "excitations", whose precise physical nature and consequences were out of reach of the biology of his day. Neuroscientists now have the knowledge and tools to tackle this question, however, and this Forum brings together leading contemporary views on the mechanisms of memory and what the engram means today.

Experience leaves a lasting structural trace in cortical circuits.

Sensory experiences exert a powerful influence on the function and future performance of neuronal circuits in the mammalian neocortex. Restructuring of synaptic connections is believed to be one mechanism by which cortical circuits store information about the sensory world. Excitatory synaptic structures, such as dendritic spines, are dynamic entities that remain sensitive to alteration of sensory input throughout life. It remains unclear, however, whether structural changes at the level of dendritic spines can outlast the original experience and thereby provide a morphological basis for long-term information storage. Here we follow spine dynamics on apical dendrites of pyramidal neurons in functionally defined regions of adult mouse visual cortex during plasticity of eye-specific responses induced by repeated closure of one eye (monocular deprivation). The first monocular deprivation episode doubled the rate of spine formation, thereby increasing spine density. This effect was specific to layer-5 cells located in binocular cortex, where most neurons increase their responsiveness to the non-deprived eye. Restoring binocular vision returned spine dynamics to baseline levels, but absolute spine density remained elevated and many monocular deprivation-induced spines persisted during this period of functional recovery. However, spine addition did not increase again when the same eye was closed for a second time. This absence of structural plasticity stands out against the robust changes of eye-specific responses that occur even faster after repeated deprivation. Thus, spines added during the first monocular deprivation experience may provide a structural basis for subsequent functional shifts. These results provide a strong link between functional plasticity and specific synaptic rearrangements, revealing a mechanism of how prior experiences could be stored in cortical circuits.

Function of dendritic spines on hippocampal inhibitory neurons.

The majority of γ-aminobutyric acid (GABA)ergic interneurons have smooth dendrites with no or only few dendritic spines, but certain types of spiny GABAergic interneurons do actually contain substantial numbers of spines. The explanation for such spines has so far been purely structural: They increase the dendritic surface area and thus provide the opportunity to accommodate larger numbers of synapses. We reasoned that there may be specific functional properties for these spines and therefore, undertook to characterize interneuron spines functionally. We find a remarkable similarity to pyramidal cell spines: They receive excitatory synapses with calcium impermeable α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors, compartmentalize biochemical signals, and display activity-dependent morphological plasticity. Nevertheless, notable differences in spine density, neck length, and spine-dendrite coupling exist. Thus, dendritic spines on inhibitory interneurons have a number of important functional properties that go substantially beyond simply expanding the dendritic surface area. It therefore seems likely that spiny and aspiny interneurons may have very different roles in neural circuit function and plasticity.

A molecular correlate of ocular dominance columns in the developing mammalian visual cortex.

Ocular dominance (OD) columns, alternating regions of left and right eye input in the visual cortex of higher mammals, have long been thought to develop from an initially intermingled state by an activity-dependent process. While indirect evidence points to potential alternative mechanisms based on molecular cues, direct proof for a molecular difference between left- and right eye columns is missing. Here, we show that heat shock protein 90 alpha (Hsp90α) is expressed in a clustered fashion in the developing cat visual cortex. Clusters of Hsp90α-positive cells are in register with OD columns of the ipsilateral eye as early as postnatal day 16, when OD columns have just appeared. Importantly, a periodic, clustered expression of Hsp90α is already present weeks before OD columns have started to form, suggesting that molecular differences between future left and right eye OD columns may contribute to the segregated termination of eye specific afferents in the developing visual cortex.

Altered visual experience induces instructive changes of orientation preference in mouse visual cortex.

Stripe rearing, the restriction of visual experience to contours of only one orientation, leads to an overrepresentation of the experienced orientation among neurons in the visual cortex. It is unclear, however, how these changes are brought about. Are they caused by silencing of neurons tuned to non-experienced orientations, or do some neurons change their preferred orientation? To address this question, we stripe-reared juvenile mice using cylinder lens goggles. Following stripe rearing, the orientation preference of cortical neurons was determined with two-photon calcium imaging. This allowed us to sample all neurons in a given field of view, including the non-responsive ones, thus overcoming a fundamental limitation of extracellular electrophysiological recordings. Stripe rearing for 3 weeks resulted in a clear overrepresentation of the experienced orientation in cortical layer 2/3. Closer inspection revealed that the stripe rearing effect changed with depth in cortex: The fraction of responsive neurons decreased in upper layer 2/3, but changed very little deeper in this layer. At the same time, the overrepresentation of the experienced orientation was strongest in lower layer 2/3. Thus, diverse mechanisms contribute to the overall stripe rearing effect, but for neurons in lower layer 2/3 the effect is mediated by an instructive mechanism, which alters the orientation tuning of individual neurons.

Doxycycline-dependent photoactivated gene expression in eukaryotic systems.

High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activation of transgenes based on the 'Tet-on' system. After incubation with caged doxycycline or caged cyanodoxycycline, we induced gene expression by local irradiation with UV light or by two-photon uncaging in diverse biological systems, including mouse organotypic brain cultures, developing mouse embryos and Xenopus laevis tadpoles. The amount of UV light needed for induction was harmless as we detected no signs of toxicity. This method allows high-resolution conditional transgene expression at different spatial scales, ranging from single cells to entire complex organisms.

Highly ordered arrangement of single neurons in orientation pinwheels.

In the visual cortex of higher mammals, neurons are arranged across the cortical surface in an orderly map of preferred stimulus orientations. This map contains 'orientation pinwheels', structures that are arranged like the spokes of a wheel such that orientation changes continuously around a centre. Conventional optical imaging first demonstrated these pinwheels, but the technique lacked the spatial resolution to determine the response properties and arrangement of cells near pinwheel centres. Electrophysiological recordings later demonstrated sharply selective neurons near pinwheel centres, but it remained unclear whether they were arranged randomly or in an orderly fashion. Here we use two-photon calcium imaging in vivo to determine the microstructure of pinwheel centres in cat visual cortex with single-cell resolution. We find that pinwheel centres are highly ordered: neurons selective to different orientations are clearly segregated even in the very centre. Thus, pinwheel centres truly represent singularities in the cortical map. This highly ordered arrangement at the level of single cells suggests great precision in the development of cortical circuits underlying orientation selectivity.

Orientation and direction tuning align with dendritic morphology and spatial connectivity in mouse visual cortex.

The functional properties of neocortical pyramidal cells (PCs), such as direction and orientation selectivity in visual cortex, predominantly derive from their excitatory and inhibitory inputs. For layer 2/3 (L2/3) PCs, the detailed relationship between their functional properties and how they sample and integrate information across cortical space is not fully understood. Here, we study this relationship by combining functional in vivo two-photon calcium imaging, in vitro functional circuit mapping, and dendritic reconstruction of the same L2/3 PCs in mouse visual cortex. Our work reveals direct correlations between dendritic morphology and functional input connectivity and the orientation as well as direction tuning of L2/3 PCs. First, the apical dendritic tree is elongated along the postsynaptic preferred orientation, considering the representation of visual space in the cortex as determined by its retinotopic organization. Additionally, sharply orientation-tuned cells show a less complex apical tree compared with broadly tuned cells. Second, in direction-selective L2/3 PCs, the spatial distribution of presynaptic partners is offset from the soma opposite to the preferred direction. Importantly, although the presynaptic excitatory and inhibitory input distributions spatially overlap on average, the excitatory input distribution is spatially skewed along the preferred direction, in contrast to the inhibitory distribution. Finally, the degree of asymmetry is positively correlated with the direction selectivity of the postsynaptic L2/3 PC. These results show that the dendritic architecture and the spatial arrangement of excitatory and inhibitory presynaptic cells of L2/3 PCs play important roles in shaping their orientation and direction tuning.

Homeostatic regulation of eye-specific responses in visual cortex during ocular dominance plasticity.

Experience-dependent plasticity is crucial for the precise formation of neuronal connections during development. It is generally thought to depend on Hebbian forms of synaptic plasticity. In addition, neurons possess other, homeostatic means of compensating for changes in sensory input, but their role in cortical plasticity is unclear. We used two-photon calcium imaging to investigate whether homeostatic response regulation contributes to changes of eye-specific responsiveness after monocular deprivation (MD) in mouse visual cortex. Short MD durations decreased deprived-eye responses in neurons with binocular input. Longer MD periods strengthened open-eye responses, and surprisingly, also increased deprived-eye responses in neurons devoid of open-eye input. These bidirectional response adjustments effectively preserved the net visual drive for each neuron. Our finding that deprived-eye responses were either weaker or stronger after MD, depending on the amount of open-eye input a cell received, argues for both Hebbian and homeostatic mechanisms regulating neuronal responsiveness during experience-dependent plasticity.

Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion.

We tracked pathogenic myelin basic protein-specific CD4+ effector T cells in early central nervous system (CNS) lesions of experimental autoimmune encephalomyelitis (EAE) by combining two-photon imaging and fluorescence video microscopy. We made two key observations: (a) the majority of the cells (65%) moved fast (maximal speed 25 microm/min) and apparently nondirected through the compact tissue; and (b) a second group of effector T cells (35%) appeared tethered to a fixed point. Polarization of T cell receptor and adhesion molecules (lymphocyte function-associated antigen 1) towards this fixed point suggests the formation of immune synapses. Nonpathogenic, ovalbumin-specific T cells were not tethered in the CNS and did not form synapse-like contacts, but moved through the tissue. After intrathecal injection of antigen, 40% of ovalbumin-specific T cells became tethered. Conversely, injection of anti-major histocompatibility complex class II antibodies profoundly reduced the number of stationary pathogenic T cells within the CNS (to 15%). We propose that rapid penetration of the CNS parenchyma by numerous autoimmune effector T cells along with multiple autoantigen-presentation events are responsible for the fulminate development of clinical EAE.