Dense Circuit Reconstruction to Understand Neuronal Computation: Focus on Zebrafish.

The dense reconstruction of neuronal wiring diagrams from volumetric electron microscopy data has the potential to generate fundamentally new insights into mechanisms of information processing and storage in neuronal circuits. Zebrafish provide unique opportunities for dynamical connectomics approaches that combine reconstructions of wiring diagrams with measurements of neuronal population activity and behavior. Such approaches have the power to reveal higher-order structure in wiring diagrams that cannot be detected by sparse sampling of connectivity and that is essential for neuronal computations. In the brain stem, recurrently connected neuronal modules were identified that can account for slow, low-dimensional dynamics in an integrator circuit. In the spinal cord, connectivity specifies functional differences between premotor interneurons. In the olfactory bulb, tuning-dependent connectivity implements a whitening transformation that is based on the selective suppression of responses to overrepresented stimulus features. These findings illustrate the potential of dynamical connectomics in zebrafish to analyze the circuit mechanisms underlying higher-order neuronal computations.

Optogenetic Inhibition of the Cortical Efferents to the Locus Ceruleus Region of Pontine Tegmentum Causes Cognitive Deficits.

BACKGROUND: The medial prefrontal cortex (mPFC) is synaptically coupled to locus ceruleus (LC) located in the pontine tegmentum. The LC supplies norepinephrine (NE) to most of the central nervous system (CNS) via an elaborate efferent network. NE release in the cortex and various limbic structures regulates arousal, memory processes, adaptive behavior and cognitive control. METHODS: The study investigated the role of the mPFC-LC circuit in the cognitive behavior of mice. The mPFC efferents were inhibited optogenetically at the level of dorso-rostral pons by virally delivered ArchT opsin. The mice were implanted bilaterally with optic fibers transmitting yellow light and tested for anxiety-like behavior on Elevated O-maze (EOM), for long-term memory with Novel Object Recognition test (NOR), for problem-solving ability with Puzzle test and for learning with Cued Fear Conditioning (FC). In addition, we used anterograde transsynaptic viral tracing to map a possible anatomical circuit allowing the mPFC to modulate the activity of LC neurons, which supply NE to the main limbic structures with a functional role in cognitive behavior. RESULTS: The application of yellow light did not affect the anxiety-like behavior of the mice but impaired their ability to recognize a novel object and solve a problem. Optogenetic inhibition of mPFC to LC, in either acquisition or recall phase of FC similarly decreased freezing. The viral tracing identified the following tripartite circuits: mPFC-LC-dentate gyrus of the hippocampus (DG), mPFC-LC-amygdala (Amy), and mPFC-LC-mPFC. CONCLUSIONS: Our results reveal essential long-range regulatory circuits from the mPFC to LC and from LC to the limbic system that serves to optimize cognitive performance.

Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

Modelling learning in Caenorhabditis elegans chemosensory and locomotive circuitry for T-maze navigation.

Recently, a new type of Caenorhabditis elegans associative learning was reported, where nematodes learn to reach a target arm in an empty T-maze, after they have successfully located reward (food) in the same side arm of a similar, baited, training maze. Here, we present a simplified mathematical model of C. elegans chemosensory and locomotive circuitry that replicates C. elegans navigation in a T-maze and predicts the underlying mechanisms generating maze learning. Based on known neural circuitry, the model circuit responds to food-released chemical cues by modulating motor neuron activity that drives simulated locomotion. We show that, through modulation of interneuron activity, such a circuit can mediate maze learning by acquiring a turning bias, even after a single training session. Simulated nematode maze navigation during training conditions in food-baited mazes and during testing conditions in empty mazes is validated by comparing simulated behaviour with new experimental video data, extracted through the implementation of a custom-made maze tracking algorithm. Our work provides a mathematical framework for investigating the neural mechanisms underlying this novel learning behaviour in C. elegans. Model results predict neuronal components involved in maze and spatial learning and identify target neurons and potential neural mechanisms for future experimental investigations into this learning behaviour.

Distinct interhemispheric connectivity at the level of the olfactory bulb emerges during Xenopus laevis metamorphosis.

During metamorphosis, the olfactory system of anuran tadpoles undergoes substantial restructuring. The main olfactory epithelium in the principal nasal cavity of Xenopus laevis tadpoles is associated with aquatic olfaction and transformed into the adult air-nose, while a new adult water-nose emerges in the middle cavity. Impacts of this metamorphic remodeling on odor processing, behavior, and network structure are still unexplored. Here, we used neuronal tracings, calcium imaging, and behavioral experiments to examine the functional connectivity between the epithelium and the main olfactory bulb during metamorphosis. In tadpoles, olfactory receptor neurons in the principal cavity project axons to glomeruli in the ventral main olfactory bulb. These projections are gradually replaced by receptor neuron axons from the newly forming middle cavity epithelium. Despite this reorganization in the ventral bulb, two spatially segregated odor processing streams remain undisrupted and behavioral responses to waterborne odorants are unchanged. Contemporaneously, new receptor neurons in the remodeling principal cavity innervate the emerging dorsal part of the bulb, which displays distinct wiring features. Glomeruli around its midline are innervated from the left and right nasal epithelia. Additionally, postsynaptic projection neurons in the dorsal bulb predominantly connect to multiple glomeruli, while half of projection neurons in the ventral bulb are uni-glomerular. Our results show that the "water system" remains functional despite metamorphic reconstruction. The network differences between the dorsal and ventral olfactory bulb imply a higher degree of odor integration in the dorsal main olfactory bulb. This is possibly connected with the processing of different odorants, airborne vs. waterborne.

Rho Guanine Nucleotide Exchange Factors Regulate Horizontal Axon Branching of Cortical Upper Layer Neurons.

Axon branching is a crucial process for cortical circuit formation. However, how the cytoskeletal changes in axon branching are regulated is not fully understood. In the present study, we investigated the role of RhoA guanine nucleotide exchange factors (RhoA-GEFs) in branch formation of horizontally elongating axons (horizontal axons) in the mammalian cortex. In situ hybridization showed that more than half of all known RhoA-GEFs were expressed in the developing rat cortex. These RhoA-GEFs were mostly expressed in the macaque cortex as well. An overexpression study using organotypic cortical slice cultures demonstrated that several RhoA-GEFs strongly promoted horizontal axon branching. Moreover, branching patterns were different between overexpressed RhoA-GEFs. In particular, ARHGEF18 markedly increased terminal arbors, whereas active breakpoint cluster region-related protein (ABR) increased short branches in both distal and proximal regions of horizontal axons. Rho kinase inhibitor treatment completely suppressed the branch-promoting effect of ARHGEF18 overexpression, but only partially affected that of ABR, suggesting that these RhoA-GEFs employ distinct downstream pathways. Furthermore, knockdown of either ARHGEF18 or ABR considerably suppressed axon branching. Taken together, the present study revealed that subsets of RhoA-GEFs differentially promote axon branching of mammalian cortical neurons.

Transplanted Embryonic Neurons Improve Functional Recovery by Increasing Activity in Injured Cortical Circuits.

Transplantation of appropriate neuronal precursors after injury is a promising strategy to reconstruct cortical circuits, but the efficiency of these approaches remains limited. Here, we applied targeted apoptosis to selectively ablate layer II/III pyramidal neurons in the rat juvenile cerebral cortex and attempted to replace lost neurons with their appropriate embryonic precursors by transplantation. We demonstrate that grafted precursors do not migrate to replace lost neurons but form vascularized clusters establishing reciprocal synaptic contacts with host networks and show functional integration. These heterotopic neuronal clusters significantly enhance the activity of the host circuits without causing epileptic seizures and attenuate the apoptotic injury-induced functional deficits in electrophysiological and behavioral tests. Chemogenetic activation of grafted neurons further improved functional recovery, and the persistence of the graft was necessary for maintaining restored functions in adult animals. Thus, implanting neuronal precursors capable to form synaptically integrated neuronal clusters combined with activation-based approaches represents a useful strategy for helping long-term functional recovery following brain injury.

Functional mosaic organization of neuroligins in neuronal circuits.

Complex brain circuitry with feedforward and feedback systems regulates neuronal activity, enabling neural networks to process and drive the entire spectrum of cognitive, behavioral, sensory, and motor functions. Simultaneous orchestration of distinct cells and interconnected neural circuits is underpinned by hundreds of synaptic adhesion molecules that span synaptic junctions. Dysfunction of a single molecule or molecular interaction at synapses can lead to disrupted circuit activity and brain disorders. Neuroligins, a family of cell adhesion molecules, were first identified as postsynaptic-binding partners of presynaptic neurexins and are essential for synapse specification and maturation. Here, we review recent advances in our understanding of how this family of adhesion molecules controls neuronal circuit assembly by acting in a synapse-specific manner.

Presynaptic development is controlled by the core active zone proteins CAST/ELKS.

KEY POINTS: CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held. CAST/ELKS regulate all three CaV 2 subtype channel levels in the presynaptic terminal and not just CaV 2.1. The half-life of ELKS is on the timescale of days and not weeks. Synaptic transmission was not impacted by the loss of CAST/ELKS. CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. ABSTRACT: Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z-stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV 2 channel subtype currents that correlated with a loss in presynaptic CaV 2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV 2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV 2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation.

Purinergic modulation of neuronal gap junction circuits in the CNS of the leech.

Gap junctions (GJs) are widely distributed in brains across the animal kingdom. To visualize the GJ- coupled networks of two major mechanosensory neurons in the ganglia of medicinal leeches, we injected these cells with the GJ-permeable tracer Neurobiotin. When diffusion time was limited to only 30 min, tracer coupling was highly variable for both cells, suggesting a possible modulation of GJ permeability. In invertebrates the innexins (homologs of vertebrate pannexins) form the GJs. Because extracellular adenosine triphosphate (ATP) modulates pannexin and leech innexin hemichannel permeability and is released by leech glial cells following injury, we tested the effects of bath application of ATP after the injection of Neurobiotin and observed a significant increase in the number of neurons tracer coupled to the sensory neurons. This effect required the elevation of intracellular Ca2+ and could be produced by bath application of caffeine. Conversely, scavenging endogenous extracellular ATP with the ATPase apyrase decreased the number of coupled cells. ATP also increased electrical conductance and tracer permeability between the bilateral Retzius neurons. This modulatory effect of ATP on GJ coupling was blocked by siRNA knockdown of a P1-like adenosine receptor. Finally, exposure of leech ganglia to extracellular ATP induced a characteristic low frequency (<0.3 Hz) rhythmic bursting activity that was roughly synchronous among multiple neurons, a behavior that was significantly attenuated by the GJ blocker octanol. These findings highlight the mediation by ATP of a robust physiological mechanism for modifying neuronal circuits by rapidly recruiting neurons into active networks and entraining synchronized bursting activity.

Cellular and circuit mechanisms of olfactory associative learning in Drosophila.

Recent years have witnessed significant progress in understanding how memories are encoded, from the molecular to the cellular and the circuit/systems levels. With a good compromise between brain complexity and behavioral sophistication, the fruit fly Drosophila melanogaster is one of the preeminent animal models of learning and memory. Here we review how memories are encoded in Drosophila, with a focus on short-term memory and an eye toward future directions. Forward genetic screens have revealed a large number of genes and transcripts necessary for learning and memory, some acting cell-autonomously. Further, the relative numerical simplicity of the fly brain has enabled the reverse engineering of learning circuits with remarkable precision, in some cases ascribing behavioral phenotypes to single neurons. Functional imaging and physiological studies have localized and parsed the plasticity that occurs during learning at some of the major loci. Connectomics projects are significantly expanding anatomical knowledge of the nervous system, filling out the roadmap for ongoing functional/physiological and behavioral studies, which are being accelerated by simultaneous tool development. These developments have provided unprecedented insight into the fundamental neural principles of learning, and lay the groundwork for deep understanding in the near future.

In the Right Place at the Right Time: miRNAs as Key Regulators in Developing Axons.

During neuronal circuit formation, axons progressively develop into a presynaptic compartment aided by extracellular signals. Axons display a remarkably high degree of autonomy supported in part by a local translation machinery that permits the subcellular production of proteins required for their development. Here, we review the latest findings showing that microRNAs (miRNAs) are critical regulators of this machinery, orchestrating the spatiotemporal regulation of local translation in response to cues. We first survey the current efforts toward unraveling the axonal miRNA repertoire through miRNA profiling, and we reveal the presence of a putative axonal miRNA signature. We also provide an overview of the molecular underpinnings of miRNA action. Our review of the available experimental evidence delineates two broad paradigms: cue-induced relief of miRNA-mediated inhibition, leading to bursts of protein translation, and cue-induced miRNA activation, which results in reduced protein production. Overall, this review highlights how a decade of intense investigation has led to a new appreciation of miRNAs as key elements of the local translation regulatory network controlling axon development.

Anterograde Neuronal Circuit Tracers Derived from Herpes Simplex Virus 1: Development, Application, and Perspectives.

Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded DNA genome provides great capacity for exogenous genes. Moreover, the features of neuron infection and neuron-to-neuron spread also offer special value to neuroscience. HSV-1 strain H129, with its predominant anterograde transneuronal transmission, represents one of the most promising anterograde neuronal circuit tracers to map output neuronal pathways. Decades of development have greatly expanded the H129-derived anterograde tracing toolbox, including polysynaptic and monosynaptic tracers with various fluorescent protein labeling. These tracers have been applied to neuroanatomical studies, and have contributed to revealing multiple important neuronal circuits. However, current H129-derived tracers retain intrinsic drawbacks that limit their broad application, such as yet-to-be improved labeling intensity, potential nonspecific retrograde labeling, and high toxicity. The biological complexity of HSV-1 and its insufficiently characterized virological properties have caused difficulties in its improvement and optimization as a viral tool. In this review, we focus on the current H129-derived viral tracers and highlight strategies in which future technological development can advance its use as a tool.

Neuropeptides as Regulators of Behavior in Insects.

Neuropeptides are by far the largest and most diverse group of signaling molecules in multicellular organisms. They are ancient molecules important in regulating a multitude of processes. Their small proteinaceous character allowed them to evolve and radiate quickly into numerous different molecules. On average, hundreds of distinct neuropeptides are present in animals, sometimes with unique classes that do not occur in distantly related species. Acting as neurotransmitters, neuromodulators, hormones, or growth factors, they are extremely diverse and are involved in controlling growth, development, ecdysis, digestion, diuresis, and many more physiological processes. Neuropeptides are also crucial in regulating myriad behavioral actions associated with feeding, courtship, sleep, learning and memory, stress, addiction, and social interactions. In general, behavior ensures that an organism can survive in its environment and is defined as any action that can change an organism's relationship to its surroundings. Even though the mode of action of neuropeptides in insects has been vigorously studied, relatively little is known about most neuropeptides and only a few model insects have been investigated. Here, we provide an overview of the roles neuropeptides play in insect behavior. We conclude that multiple neuropeptides need to work in concert to coordinate certain behaviors. Additionally, most neuropeptides studied to date have more than a single function.

Does Global Astrocytic Calcium Signaling Participate in Awake Brain State Transitions and Neuronal Circuit Function?

We continuously need to adapt to changing conditions within our surrounding environment, and our brain needs to quickly shift between resting and working activity states in order to allow appropriate behaviors. These global state shifts are intimately linked to the brain-wide release of the neuromodulators, noradrenaline and acetylcholine. Astrocytes have emerged as a new player participating in the regulation of brain activity, and have recently been implicated in brain state shifts. Astrocytes display global Ca2+ signaling in response to activation of the noradrenergic system, but whether astrocytic Ca2+ signaling is causative or correlative for shifts in brain state and neural activity patterns is not known. Here we review the current available literature on astrocytic Ca2+ signaling in awake animals in order to explore the role of astrocytic signaling in brain state shifts. Furthermore, we look at the development and availability of innovative new methodological tools that are opening up for new ways of visualizing and perturbing astrocyte activity in awake behaving animals. With these new tools at hand, the field of astrocyte research will likely be able to elucidate the causal and mechanistic roles of astrocytes in complex behaviors within a very near future.

Consistent estimation of complete neuronal connectivity in large neuronal populations using sparse "shotgun" neuronal activity sampling.

We investigate the properties of recently proposed "shotgun" sampling approach for the common inputs problem in the functional estimation of neuronal connectivity. We study the asymptotic correctness, the speed of convergence, and the data size requirements of such an approach. We show that the shotgun approach can be expected to allow the inference of complete connectivity matrix in large neuronal populations under some rather general conditions. However, we find that the posterior error of the shotgun connectivity estimator grows quickly with the size of unobserved neuronal populations, the square of average connectivity strength, and the square of observation sparseness. This implies that the shotgun connectivity estimation will require significantly larger amounts of neuronal activity data whenever the number of neurons in observed neuronal populations remains small. We present a numerical approach for solving the shotgun estimation problem in general settings and use it to demonstrate the shotgun connectivity inference in the examples of simulated synfire and weakly coupled cortical neuronal networks.

Segregated labeling of olfactory bulb projection neurons based on their birthdates.

Mitral and tufted cells are the projection neurons of the olfactory bulb (OB). We previously reported that somata location and innervation patterns were different between early- and late-born mitral cells (Imamura et al., 2011). Here, we introduced a plasmid that drives the expression of a GFP gene into the mouse OB using in utero electroporation, and demonstrated that we can deliver the plasmid vectors into distinct subsets of OB projection neurons by changing the timing of electroporation after fertilisation. The electroporation performed at embryonic day (E)10 preferentially labeled mitral cells in the accessory OB and main OB mitral cells in dorsomedial mitral cell layer (MCL). In contrast, the E12 electroporation introduced the plasmid vectors preferentially into main OB mitral cells in the ventrolateral MCL and tufted cells. Combining these data with BrdU injections, we confirmed that E10 and E12 electroporation preferentially labeled early- and late-born projection neurons, respectively. This work introduces a novel method for segregated labeling of mouse olfactory bulb projection neurons based on their birthdates. With this technique we found that early- and late-born projection neurons extend their secondary dendrites in the deep and superficial external plexiform layer (EPL), respectively. Although a similar segregation has been suggested for mitral vs. tufted cell dendrites, we found mitral cells projecting secondary dendrites into the superficial EPL in E12-electroporated main OB. Our observations indicate that timing of neurogenesis regulates not only somata location and innervation patterns but also the laminar organisation of projection neuron dendrites in the EPL.

Neuronal Circuit Dysfunction in Amyotrophic Lateral Sclerosis.

The primary neural circuit affected in Amyotrophic Lateral Sclerosis (ALS) patients is the corticospinal motor circuit, originating in upper motor neurons (UMNs) in the cerebral motor cortex which descend to synapse with the lower motor neurons (LMNs) in the spinal cord to ultimately innervate the skeletal muscle. Perturbation of these neural circuits and consequent loss of both UMNs and LMNs, leading to muscle wastage and impaired movement, is the key pathophysiology observed. Despite decades of research, we are still lacking in ALS disease-modifying treatments. In this review, we document the current research from patient studies, rodent models, and human stem cell models in understanding the mechanisms of corticomotor circuit dysfunction and its implication in ALS. We summarize the current knowledge about cortical UMN dysfunction and degeneration, altered excitability in LMNs, neuromuscular junction degeneration, and the non-cell autonomous role of glial cells in motor circuit dysfunction in relation to ALS. We further highlight the advances in human stem cell technology to model the complex neural circuitry and how these can aid in future studies to better understand the mechanisms of neural circuit dysfunction underpinning ALS.

Transient sensorimotor projections in the developmental song learning period.

Memory recall and guidance are essential for motor skill acquisition. Like humans learning to speak, male zebra finches learn to sing by first memorizing and then matching their vocalization to the tutor's song (TS) during specific developmental periods. Yet, the neuroanatomical substrate supporting auditory-memory-guided sensorimotor learning has remained elusive. Here, using a whole-brain connectome analysis with activity-dependent viral expression, we identified a transient projection into the motor region, HVC, from neuronal ensembles responding to TS in the auditory forebrain, the caudomedial nidopallium (NCM), in juveniles. Virally induced cell death of the juvenile, but not adult, TS-responsive NCM neurons impaired song learning. Moreover, isolation, which delays closure of the sensory, but not the motor, learning period, did not affect the decrease of projections into the HVC from the NCM TS-responsive neurons after the song learning period. Taken together, our results suggest that dynamic axonal pruning may regulate timely auditory-memory-guided vocal learning during development.

Axon guidance during mouse central nervous system regeneration is required for specific brain innervation.

Reconstructing functional neuronal circuits is one major challenge of central nervous system repair. Through activation of pro-growth signaling pathways, some neurons achieve long-distance axon regrowth. Yet, functional reconnection has hardly been obtained, as these regenerating axons fail to resume their initial trajectory and reinnervate their proper target. Axon guidance is considered to be active only during development. Here, using the mouse visual system, we show that axon guidance is still active in the adult brain in regenerative conditions. We highlight that regenerating retinal ganglion cell axons avoid one of their primary targets, the suprachiasmatic nucleus (SCN), due to Slit/Robo repulsive signaling. Together with promoting regeneration, silencing Slit/Robo in vivo enables regenerating axons to enter the SCN and form active synapses. The newly formed circuit is associated with neuronal activation and functional recovery. Our results provide evidence that axon guidance mechanisms are required to reconnect regenerating axons to specific brain nuclei.