Slow kinesin-dependent microtubular transport facilitates ribbon synapse assembly in developing cochlear inner hair cells.

Sensory synapses are characterized by electron-dense presynaptic specializations, so-called synaptic ribbons. In cochlear inner hair cells (IHCs), ribbons play an essential role as core active zone (AZ) organizers, where they tether synaptic vesicles, cluster calcium channels and facilitate the temporally-precise release of primed vesicles. While a multitude of studies aimed to elucidate the molecular composition and function of IHC ribbon synapses, the developmental formation of these signalling complexes remains largely elusive to date. To address this shortcoming, we performed long-term live-cell imaging of fluorescently-labelled ribbon precursors in young postnatal IHCs to track ribbon precursor motion. We show that ribbon precursors utilize the apico-basal microtubular (MT) cytoskeleton for targeted trafficking to the presynapse, in a process reminiscent of slow axonal transport in neurons. During translocation, precursor volume regulation is achieved by highly dynamic structural plasticity - characterized by regularly-occurring fusion and fission events. Pharmacological MT destabilization negatively impacted on precursor translocation and attenuated structural plasticity, whereas genetic disruption of the anterograde molecular motor Kif1a impaired ribbon volume accumulation during developmental maturation. Combined, our data thus indicate an essential role of the MT cytoskeleton and Kif1a in adequate ribbon synapse formation and structural maintenance.

Dynamic Gain Decomposition Reveals Functional Effects of Dendrites, Ion Channels, and Input Statistics in Population Coding.

Modern, high-density neuronal recordings reveal at ever higher precision how information is represented by neural populations. Still, we lack the tools to understand these processes bottom-up, emerging from the biophysical properties of neurons, synapses, and network structure. The concept of the dynamic gain function, a spectrally resolved approximation of a population's coding capability, has the potential to link cell-level properties to network-level performance. However, the concept is not only useful but also very complex because the dynamic gain's shape is co-determined by axonal and somato-dendritic parameters and the population's operating regime. Previously, this complexity precluded an understanding of any individual parameter's impact. Here, we decomposed the dynamic gain function into three components corresponding to separate signal transformations. This allowed attribution of network-level encoding features to specific cell-level parameters. Applying the method to data from real neurons and biophysically plausible models, we found: (1) The encoding bandwidth of real neurons, approximately 400 Hz, is constrained by the voltage dependence of axonal currents during early action potential initiation. (2) State-of-the-art models only achieve encoding bandwidths around 100 Hz and are limited mainly by subthreshold processes instead. (3) Large dendrites and low-threshold potassium currents modulate the bandwidth by shaping the subthreshold stimulus-to-voltage transformation. Our decomposition provides physiological interpretations when the dynamic gain curve changes, for instance during spectrinopathies and neurodegeneration. By pinpointing shortcomings of current models, it also guides inference of neuron models best suited for large-scale network simulations.

Dynamics and functions of E-cadherin complexes in epithelial cell and tissue morphogenesis.

Cell-cell adhesion is at the center of structure and dynamics of epithelial tissue. E-cadherin-catenin complexes mediate Ca2+-dependent trans-homodimerization and constitute the kernel of adherens junctions. Beyond the basic function of cell-cell adhesion, recent progress sheds light the dynamics and interwind interactions of individual E-cadherin-catenin complex with E-cadherin superclusters, contractile actomyosin and mechanics of the cortex and adhesion. The nanoscale architecture of E-cadherin complexes together with cis-interactions and interactions with cortical actomyosin adjust to junctional tension and mechano-transduction by reinforcement or weakening of specific features of the interactions. Although post-translational modifications such as phosphorylation and glycosylation have been implicated, their role for specific aspects of in E-cadherin function has remained unclear. Here, we provide an overview of the E-cadherin complex in epithelial cell and tissue morphogenesis focusing on nanoscale architectures by super-resolution approaches and post-translational modifications from recent, in particular in vivo, studies. Furthermore, we review the computational modelling in E-cadherin complexes and highlight how computational modelling has contributed to a deeper understanding of the E-cadherin complexes.

Fibroblast growth factor 9 (FGF9)-mediated neurodegeneration: Implications for progressive multiple sclerosis?

AIMS: Fibroblast growth factor (FGF) signalling is dysregulated in multiple sclerosis (MS) and other neurological and psychiatric conditions, but there is little or no consensus as to how individual FGF family members contribute to disease pathogenesis. Lesion development in MS is associated with increased expression of FGF1, FGF2 and FGF9, all of which modulate remyelination in a variety of experimental settings. However, FGF9 is also selectively upregulated in major depressive disorder (MDD), prompting us to speculate it may also have a direct effect on neuronal function and survival. METHODS: Transcriptional profiling of myelinating cultures treated with FGF1, FGF2 or FGF9 was performed, and the effects of FGF9 on cortical neurons investigated using a combination of transcriptional, electrophysiological and immunofluorescence microscopic techniques. The in vivo effects of FGF9 were explored by stereotactic injection of adeno-associated viral (AAV) vectors encoding either FGF9 or EGFP into the rat motor cortex. RESULTS: Transcriptional profiling of myelinating cultures after FGF9 treatment revealed a distinct neuronal response with a pronounced downregulation of gene networks associated with axonal transport and synaptic function. In cortical neuronal cultures, FGF9 also rapidly downregulated expression of genes associated with synaptic function. This was associated with a complete block in the development of photo-inducible spiking activity, as demonstrated using multi-electrode recordings of channel rhodopsin-transfected rat cortical neurons in vitro and, ultimately, neuronal cell death. Overexpression of FGF9 in vivo resulted in rapid loss of neurons and subsequent development of chronic grey matter lesions with neuroaxonal reduction and ensuing myelin loss. CONCLUSIONS: These observations identify overexpression of FGF9 as a mechanism by which neuroaxonal pathology could develop independently of immune-mediated demyelination in MS. We suggest targeting neuronal FGF9-dependent pathways may provide a novel strategy to slow if not halt neuroaxonal atrophy and loss in MS, MDD and potentially other neurodegenerative diseases.

Morphogenesis: Unstable rods and how genetics tames them.

Rods under mechanical stress are a classic example of dynamic instability. Axis elongation in Drosophila usually leads to a U-shaped axis, but folded or twisted axes are observed in certain mutants. Analysis of these mutants now reveals the source of the instability and the mechanism for maintaining left-right symmetry.

Alcohol sensitivity and tolerance encoding in sleep regulatory circadian neurons in Drosophila.

Alcohol tolerance is a simple form of behavioural and neural plasticity that occurs with the first drink. Neural plasticity in tolerance is likely a substrate for longer term adaptations that can lead to alcohol use disorder. Drosophila develop tolerance with characteristics similar to vertebrates, and it is a useful model for determining the molecular and circuit encoding mechanisms in detail. Rapid tolerance, measured after the first alcohol exposure is completely metabolized, is localized to specific brain regions that are not interconnected in an obvious way. We used a forward neuroanatomical screen to identify three new neural sites for rapid tolerance encoding. One of these was composed of two groups of neurons, the DN1a and DN1p glutamatergic neurons, that are part of the Drosophila circadian clock. We localized rapid tolerance to the two DN1a neurons that regulate arousal by light at night, temperature-dependent sleep timing, and night-time sleep. Two clock neurons that regulate evening activity, LNd6 and the 5th LNv, are postsynaptic to the DN1as, and they promote rapid tolerance via the metabotropic glutamate receptor. Thus, rapid tolerance to alcohol overlaps with sleep regulatory neural circuitry, suggesting a mechanistic link.

Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins.

During embryonic development, dramatic cell shape changes and movements reshape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by mechanosensitive multiprotein complexes assembled via multivalent connections. Here we combine genetic, cell biological, and modeling approaches to define the mechanism of action and functions of an important player, Drosophila polychaetoid, homologue of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction-cytoskeletal interface, suggesting different proteins localize and function in distinct ways, perhaps in distinct subcomplexes, but combine to produce robust connections.

Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins.

During embryonic development dramatic cell shape changes and movements re-shape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by a mechanosensitive multiprotein complex assembled via multivalent connections. Here we combine genetic, cell biological and modeling approaches to define the mechanism of action and functions of an important player, Drosophila Polychaetoid, homolog of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction-cytoskeletal interface, suggesting different proteins localize and function in distinct ways but combine to produce robust connections.

Alcohol tolerance encoding in sleep regulatory circadian neurons in Drosophila.

Alcohol tolerance is a simple form of behavioral and neural plasticity that occurs with the first drink. Neural plasticity in tolerance is likely a substrate for longer term adaptations that can lead to alcohol use disorder. Drosophila develop tolerance with characteristics similar to vertebrates, and it is useful model for determining the molecular and circuit encoding mechanisms in detail. Rapid tolerance, measured after the first alcohol exposure is completely metabolized, is localized to specific brain regions that are not interconnected in an obvious way. We used a forward neuroanatomical screen to identify three new neural sites for rapid tolerance encoding. One of these was comprised of two groups of neurons, the DN1a and DN1p glutamatergic neurons, that are part of the Drosophila circadian clock. We localized rapid tolerance to the two DN1a neurons that regulate arousal by light at night, temperature-dependent sleep timing, and night-time sleep. Two clock neurons that regulate evening activity, LNd6 and the 5th LNv, are postsynaptic to the DN1as and they promote rapid tolerance via the metabotropic glutamate receptor. Thus, rapid tolerance to alcohol overlaps with sleep regulatory neural circuitry, suggesting a mechanistic link.

Rapid and Chronic Ethanol Tolerance Are Composed of Distinct Memory-Like States in Drosophila.

Ethanol tolerance is the first type of behavioral plasticity and neural plasticity that is induced by ethanol intake, and yet its molecular and circuit bases remain largely unexplored. Here, we characterize the following three distinct forms of ethanol tolerance in male Drosophila: rapid, chronic, and repeated. Rapid tolerance is composed of two short-lived memory-like states, one that is labile and one that is consolidated. Chronic tolerance, induced by continuous exposure, lasts for 2 d, induces ethanol preference, and hinders the development of rapid tolerance through the activity of histone deacetylases (HDACs). Unlike rapid tolerance, chronic tolerance is independent of the immediate early gene Hr38/Nr4a Chronic tolerance is suppressed by the sirtuin HDAC Sirt1, whereas rapid tolerance is enhanced by Sirt1 Moreover, rapid and chronic tolerance map to anatomically distinct regions of the mushroom body learning and memory centers. Chronic tolerance, like long-term memory, is dependent on new protein synthesis and it induces the kayak/c-fos immediate early gene, but it depends on CREB signaling outside the mushroom bodies, and it does not require the Radish GTPase. Thus, chronic ethanol exposure creates an ethanol-specific memory-like state that is molecularly and anatomically different from other forms of ethanol tolerance.SIGNIFICANCE STATEMENT The pattern and concentration of initial ethanol exposure causes operationally distinct types of ethanol tolerance to form. We identify separate molecular and neural circuit mechanisms for two forms of ethanol tolerance, rapid and chronic. We also discover that chronic tolerance forms an ethanol-specific long-term memory-like state that localizes to learning and memory circuits, but it is different from appetitive and aversive long-term memories. By contrast, rapid tolerance is composed of labile and consolidated short-term memory-like states. The multiple forms of ethanol memory-like states are genetically tractable for understanding how initial forms of ethanol-induced neural plasticity form a substrate for the longer-term brain changes associated with alcohol use disorder.

Environmental enrichment enhances patterning and remodeling of synaptic nanoarchitecture as revealed by STED nanoscopy.

Synaptic plasticity underlies long-lasting structural and functional changes to brain circuitry and its experience-dependent remodeling can be fundamentally enhanced by environmental enrichment. It is however unknown, whether and how the environmental enrichment alters the morphology and dynamics of individual synapses. Here, we present a virtually crosstalk-free two-color in vivo stimulated emission depletion (STED) microscope to simultaneously superresolve the dynamics of endogenous PSD95 of the post-synaptic density and spine geometry in the mouse cortex. In general, the spine head geometry and PSD95 assemblies were highly dynamic, their changes depended linearly on their original size but correlated only mildly. With environmental enrichment, the size distributions of PSD95 and spine head sizes were sharper than in controls, indicating that synaptic strength is set more uniformly. The topography of the PSD95 nanoorganization was more dynamic after environmental enrichment; changes in size were smaller but more correlated than in mice housed in standard cages. Thus, two-color in vivo time-lapse imaging of synaptic nanoorganization uncovers a unique synaptic nanoplasticity associated with the enhanced learning capabilities under environmental enrichment.

Ultrafast population coding and axo-somatic compartmentalization.

Populations of cortical neurons respond to common input within a millisecond. Morphological features and active ion channel properties were suggested to contribute to this astonishing processing speed. Here we report an exhaustive study of ultrafast population coding for varying axon initial segment (AIS) location, soma size, and axonal current properties. In particular, we studied their impact on two experimentally observed features 1) precise action potential timing, manifested in a wide-bandwidth dynamic gain, and 2) high-frequency boost under slowly fluctuating correlated input. While the density of axonal channels and their distance from the soma had a very small impact on bandwidth, it could be moderately improved by increasing soma size. When the voltage sensitivity of axonal currents was increased we observed ultrafast coding and high-frequency boost. We conclude that these computationally relevant features are strongly dependent on axonal ion channels' voltage sensitivity, but not their number or exact location. We point out that ion channel properties, unlike dendrite size, can undergo rapid physiological modification, suggesting that the temporal accuracy of neuronal population encoding could be dynamically regulated. Our results are in line with recent experimental findings in AIS pathologies and establish a framework to study structure-function relations in AIS molecular design.

Theta activity paradoxically boosts gamma and ripple frequency sensitivity in prefrontal interneurons.

Fast oscillations in cortical circuits critically depend on GABAergic interneurons. Which interneuron types and populations can drive different cortical rhythms, however, remains unresolved and may depend on brain state. Here, we measured the sensitivity of different GABAergic interneurons in prefrontal cortex under conditions mimicking distinct brain states. While fast-spiking neurons always exhibited a wide bandwidth of around 400 Hz, the response properties of spike-frequency adapting interneurons switched with the background input's statistics. Slowly fluctuating background activity, as typical for sleep or quiet wakefulness, dramatically boosted the neurons' sensitivity to gamma and ripple frequencies. We developed a time-resolved dynamic gain analysis and revealed rapid sensitivity modulations that enable neurons to periodically boost gamma oscillations and ripples during specific phases of ongoing low-frequency oscillations. This mechanism predicts these prefrontal interneurons to be exquisitely sensitive to high-frequency ripples, especially during brain states characterized by slow rhythms, and to contribute substantially to theta-gamma cross-frequency coupling.

Punctuated evolution of visual cortical circuits? Evidence from the large rodent Dasyprocta leporina, and the tiny primate Microcebus murinus.

Recent reports of the lack of periodic orientation columns in a very large rodent species, the red-rumped agouti, and the existence of incompressible hypercolumns in the lineage of primates, as demonstrated in one of the smallest primates, the mouse lemur, strengthen the interpretation that salt-and-pepper and columns-and-pinwheel mosaics are two distinct functional layouts. These layouts do neither depend on lifestyle nor scale with body size, brain size, absolute neuron numbers, binocular overlap, or visual acuity, but are primarily distinguishable by phylogenetic traits. The predictive value of other biological signatures such as V1 neuronal surface density and the central-peripheral density ratio of retinal ganglion cells are reconsidered, and experiments elucidating the intracortical connectivity in rodents are proposed.

Stable but not rigid: Chronic in vivo STED nanoscopy reveals extensive remodeling of spines, indicating multiple drivers of plasticity.

Excitatory synapses on dendritic spines of pyramidal neurons are considered a central memory locus. To foster both continuous adaption and the storage of long-term information, spines need to be plastic and stable at the same time. Here, we advanced in vivo STED nanoscopy to superresolve distinct features of spines (head size and neck length/width) in mouse neocortex for up to 1 month. While LTP-dependent changes predict highly correlated modifications of spine geometry, we find both, uncorrelated and correlated dynamics, indicating multiple independent drivers of spine remodeling. The magnitude of this remodeling suggests substantial fluctuations in synaptic strength. Despite this high degree of volatility, all spine features exhibit persistent components that are maintained over long periods of time. Furthermore, chronic nanoscopy uncovers structural alterations in the cortex of a mouse model of neurodegeneration. Thus, at the nanoscale, stable dendritic spines exhibit a delicate balance of stability and volatility.

Thirst interneurons that promote water seeking and limit feeding behavior in Drosophila.

Thirst is a motivational state that drives behaviors to obtain water for fluid homeostasis. We identified two types of central brain interneurons that regulate thirsty water seeking in Drosophila, that we term the Janu neurons. Janu-GABA, a local interneuron in the subesophageal zone, is activated by water deprivation and is specific to thirsty seeking. Janu-AstA projects from the subesophageal zone to the superior medial protocerebrum, a higher order processing area. Janu-AstA signals with the neuropeptide Allatostatin A to promote water seeking and to inhibit feeding behavior. NPF (Drosophila NPY) neurons are postsynaptic to Janu-AstA for water seeking and feeding through the AstA-R2 galanin-like receptor. NPF neurons use NPF to regulate thirst and hunger behaviors. Flies choose Janu neuron activation, suggesting that thirsty seeking up a humidity gradient is rewarding. These findings identify novel central brain circuit elements that coordinate internal state drives to selectively control motivated seeking behavior.

Phagocyte-mediated synapse removal in cortical neuroinflammation is promoted by local calcium accumulation.

Cortical pathology contributes to chronic cognitive impairment of patients suffering from the neuroinflammatory disease multiple sclerosis (MS). How such gray matter inflammation affects neuronal structure and function is not well understood. In the present study, we use functional and structural in vivo imaging in a mouse model of cortical MS to demonstrate that bouts of cortical inflammation disrupt cortical circuit activity coincident with a widespread, but transient, loss of dendritic spines. Spines destined for removal show local calcium accumulations and are subsequently removed by invading macrophages or activated microglia. Targeting phagocyte activation with a new antagonist of the colony-stimulating factor 1 receptor prevents cortical synapse loss. Overall, our study identifies synapse loss as a key pathological feature of inflammatory gray matter lesions that is amenable to immunomodulatory therapy.

Orientation Preference Maps in Microcebus murinus Reveal Size-Invariant Design Principles in Primate Visual Cortex.

Orientation preference maps (OPMs) are a prominent feature of primary visual cortex (V1) organization in many primates and carnivores. In rodents, neurons are not organized in OPMs but are instead interspersed in a "salt and pepper" fashion, although clusters of orientation-selective neurons have been reported. Does this fundamental difference reflect the existence of a lower size limit for orientation columns (OCs) below which they cannot be scaled down with decreasing V1 size? To address this question, we examined V1 of one of the smallest living primates, the 60-g prosimian mouse lemur (Microcebus murinus). Using chronic intrinsic signal imaging, we found that mouse lemur V1 contains robust OCs, which are arranged in a pinwheel-like fashion. OC size in mouse lemurs was found to be only marginally smaller compared to the macaque, suggesting that these circuit elements are nearly incompressible. The spatial arrangement of pinwheels is well described by a common mathematical design of primate V1 circuit organization. In order to accommodate OPMs, we found that the mouse lemur V1 covers one-fifth of the cortical surface, which is one of the largest V1-to-cortex ratios found in primates. These results indicate that the primate-type visual cortical circuit organization is constrained by a size limitation and raises the possibility that its emergence might have evolved by disruptive innovation rather than gradual change.

Spatial clustering of orientation preference in primary visual cortex of the large rodent agouti.

All rodents investigated so far possess orientation-selective neurons in the primary visual cortex (V1) but - in contrast to carnivores and primates - no evidence of periodic maps with pinwheel-like structures. Theoretical studies debating whether phylogeny or universal principles determine development of pinwheels point to V1 size as a critical constraint. Thus, we set out to study maps of agouti, a big diurnal rodent with a V1 size comparable to cats'. In electrophysiology, we detected interspersed orientation and direction-selective neurons with a bias for horizontal contours, corroborated by homogeneous activation in optical imaging. Compatible with spatial clustering at short distance, nearby neurons tended to exhibit similar orientation preference. Our results argue against V1 size as a key parameter in determining the presence of periodic orientation maps. They are consistent with a phylogenetic influence on the map layout and development, potentially reflecting distinct retinal traits or interspecies differences in cortical circuitry.