Brain wiring determinants uncovered by integrating connectomes and transcriptomes.

Advances in brain connectomics have demonstrated the extraordinary complexity of neural circuits.1,2,3,4,5 Developing neurons encounter the axons and dendrites of many different neuron types and form synapses with only a subset of them. During circuit assembly, neurons express cell-type-specific repertoires comprising many cell adhesion molecules (CAMs) that can mediate interactions between developing neurites.6,7,8 Many CAM families have been shown to contribute to brain wiring in different ways.9,10 It has been challenging, however, to identify receptor-ligand pairs directly matching neurons with their synaptic targets. Here, we integrated the synapse-level connectome of the neural circuit11,12 with the developmental expression patterns7 and binding specificities of CAMs6,13 on pre- and postsynaptic neurons in the Drosophila visual system. To overcome the complexity of neural circuits, we focus on pairs of genetically related neurons that make differential wiring choices. In the motion detection circuit,14 closely related subtypes of T4/T5 neurons choose between alternative synaptic targets in adjacent layers of neuropil.12 This choice correlates with the matching expression in synaptic partners of different receptor-ligand pairs of the Beat and Side families of CAMs. Genetic analysis demonstrated that presynaptic Side-II and postsynaptic Beat-VI restrict synaptic partners to the same layer. Removal of this receptor-ligand pair disrupts layers and leads to inappropriate targeting of presynaptic sites and postsynaptic dendrites. We propose that different Side/Beat receptor-ligand pairs collaborate with other recognition molecules to determine wiring specificities in the fly brain. Combining transcriptomes, connectomes, and protein interactome maps allow unbiased identification of determinants of brain wiring.

Cerebral cortical regions always connect with each other via the shortest paths.

In human society, the choice of transportation mode between two cities is largely influenced by the distance between the regions. Similarly, when neurons communicate with each other within the cerebral cortex, do they establish their connections based on their physical distance? In this study, we employed a data-driven approach to explore the relationships between fiber length and corresponding geodesic distance between the fiber's two endpoints on brain surface. Diffusion-MRI-derived fiber streamlines were used to represent extra-cortical axonal connections between neurons or cortical regions, while geodesic paths between cortical points were employed to simulate intra-cortical connections. The results demonstrated that the geodesic distance between two cortical regions connected by a fiber streamline was greater than the fiber length most of the time, indicating that cortical regions tend to choose the shortest path for connection; whether it be an intra-cortical or extra-cortical route, especially when intra-cortical routes within cortical regions are longer than potential extrinsic fiber routes, there is an increased probability to establish fiber routes to connect the both regions. These findings were validated in a group of human brains and may provide insights into the underlying mechanisms of neuronal growth, connection, and wiring.

Temporal control of neuronal wiring.

Wiring an animal brain is a complex process involving a staggering number of cell-types born at different times and locations in the developing brain. Incorporation of these cells into precise circuits with high fidelity is critical for animal survival and behavior. Assembly of neuronal circuits is heavily dependent upon proper timing of wiring programs, requiring neurons to express specific sets of genes (sometimes transiently) at the right time in development. While cell-type specificity of genetic programs regulating wiring has been studied in detail, mechanisms regulating proper timing and coordination of these programs across cell-types are only just beginning to emerge. In this review, we discuss some temporal regulators of wiring programs and how their activity is controlled over time and space. A common feature emerges from these temporal regulators - they are induced by cell-extrinsic cues and control transcription factors capable of regulating a highly cell-type specific set of target genes. Target specificity in these contexts comes from cell-type specific transcription factors. We propose that the spatiotemporal specificity of wiring programs is controlled by the combinatorial activity of temporal programs and cell-type specific transcription factors. Going forward, a better understanding of temporal regulators will be key to understanding the mechanisms underlying brain wiring, and will be critical for the development of in vitro models like brain organoids.

KIF2A deficiency causes early-onset neurodegeneration.

KIF2A is an atypical kinesin that has the capacity to depolymerize microtubules. Patients carrying mutations in KIF2A suffer from progressive microcephaly and mental disabilities. While the role of this protein is well documented in neuronal migration, the relationship between its dysfunction and the pathobiology of brain disorders is unclear. Here, we report that KIF2A is dispensable for embryogenic neurogenesis but critical in postnatal stages for maturation, connectivity, and maintenance of neurons. We used a conditional approach to inactivate KIF2A in cortical progenitors, nascent postmitotic neurons, and mature neurons in mice. We show that the lack of KIF2A alters microtubule dynamics and disrupts several microtubule-dependent processes, including neuronal polarity, neuritogenesis, synaptogenesis, and axonal transport. KIF2A-deficient neurons exhibit aberrant electrophysiological characteristics, neuronal connectivity, and function, leading to their loss. The role of KIF2A is not limited to development, as fully mature neurons require KIF2A for survival. Our results emphasize an additional function of KIF2A and help explain how its mutations lead to brain disorders.

Microglia-dependent remodeling of neuronal circuits.

Microglia are tissue-resident macrophages responsible for the surveillance, neuronal support, and immune defense of the brain parenchyma. Recently, the role played by microglia in the formation and function of neuronal circuits has garnered substantial attention. During development, microglia have been shown to engulf neuronal precursors and participate in pruning mechanisms while, in the mature brain, they influence synaptic signaling, provide trophic support and shape synaptic plasticity. Recently, studies have unveiled different microglial characteristics associated with specific brain regions. This emerging view suggests that the maturation and function of distinct neuronal circuits may be potentially associated with the molecular identity microglia adopts across the brain. Here, we review and summarize the known role of these cells in the thalamus, hippocampus, cortex, and cerebellum. We focus on in vivo studies to highlight the characteristics of microglia that may be important in the remodeling of these neuronal circuits and in relation to neurodevelopmental and neuropsychiatric disorders.

Brain connectivity inversely scales with developmental temperature in Drosophila.

Variability of synapse numbers and partners despite identical genes reveals the limits of genetic determinism. Here, we use developmental temperature as a non-genetic perturbation to study variability of brain wiring and behavior in Drosophila. Unexpectedly, slower development at lower temperatures increases axo-dendritic branching, synapse numbers, and non-canonical synaptic partnerships of various neurons, while maintaining robust ratios of canonical synapses. Using R7 photoreceptors as a model, we show that changing the relative availability of synaptic partners using a DIPγ mutant that ablates R7's preferred partner leads to temperature-dependent recruitment of non-canonical partners to reach normal synapse numbers. Hence, R7 synaptic specificity is not absolute but based on the relative availability of postsynaptic partners and presynaptic control of synapse numbers. Behaviorally, movement precision is temperature robust, while movement activity is optimized for the developmentally encountered temperature. These findings suggest genetically encoded relative and scalable synapse formation to develop functional, but not identical, brains and behaviors.

Primate neuronal connections are sparse in cortex as compared to mouse.

Detailing how primate and mouse neurons differ is critical for creating generalized models of how neurons process information. We reconstruct 15,748 synapses in adult Rhesus macaques and mice and ask how connectivity differs on identified cell types in layer 2/3 of primary visual cortex. Primate excitatory and inhibitory neurons receive 2-5 times fewer excitatory and inhibitory synapses than similar mouse neurons. Primate excitatory neurons have lower excitatory-to-inhibitory (E/I) ratios than mouse but similar E/I ratios in inhibitory neurons. In both species, properties of inhibitory axons such as synapse size and frequency are unchanged, and inhibitory innervation of excitatory neurons is local and specific. Using artificial recurrent neural networks (RNNs) optimized for different cognitive tasks, we find that penalizing networks for creating and maintaining synapses, as opposed to neuronal firing, reduces the number of connections per node as the number of nodes increases, similar to primate neurons compared with mice.

From whole organism to ultrastructure: progress in axonal imaging for decoding circuit development.

Since the pioneering work of Ramón y Cajal, scientists have sought to unravel the complexities of axon development underlying neural circuit formation. Micrometer-scale axonal growth cones navigate to targets that are often centimeters away. To reach their targets, growth cones react to dynamic environmental cues that change in the order of seconds to days. Proper axon growth and guidance are essential to circuit formation, and progress in imaging has been integral to studying these processes. In particular, advances in high- and super-resolution microscopy provide the spatial and temporal resolution required for studying developing axons. In this Review, we describe how improved microscopy has revolutionized our understanding of axonal development. We discuss how novel technologies, specifically light-sheet and super-resolution microscopy, led to new discoveries at the cellular scale by imaging axon outgrowth and circuit wiring with extreme precision. We next examine how advanced microscopy broadened our understanding of the subcellular dynamics driving axon growth and guidance. We finally assess the current challenges that the field of axonal biology still faces for imaging axons, and examine how future technology could meet these needs.

The Organization of Frontostriatal Brain Wiring in Healthy Subjects Using a Novel Diffusion Imaging Fiber Cluster Analysis.

To assess normal organization of frontostriatal brain wiring, we analyzed diffusion magnetic resonance imaging (dMRI) scans in 100 young adult healthy subjects (HSs). We identified fiber clusters intersecting the frontal cortex and caudate, a core component of associative striatum, and quantified their degree of deviation from a strictly topographic pattern. Using whole brain dMRI tractography and an automated tract parcellation clustering method, we extracted 17 white matter fiber clusters per hemisphere connecting the frontal cortex and caudate. In a novel approach to quantify the geometric relationship among clusters, we measured intercluster endpoint distances between corresponding cluster pairs in the frontal cortex and caudate. We show first, the overall frontal cortex wiring pattern of the caudate deviates from a strictly topographic organization due to significantly greater convergence in regionally specific clusters; second, these significantly convergent clusters originate in subregions of ventrolateral, dorsolateral, and orbitofrontal prefrontal cortex (PFC); and, third, a similar organization in both hemispheres. Using a novel tractography method, we find PFC-caudate brain wiring in HSs deviates from a strictly topographic organization due to a regionally specific pattern of cluster convergence. We conjecture cortical subregions projecting to the caudate with greater convergence subserve functions that benefit from greater circuit integration.

Miswiring of Frontostriatal Projections in Schizophrenia.

We investigated brain wiring in chronic schizophrenia and healthy controls in frontostriatal circuits using diffusion magnetic resonance imaging tractography in a novel way. We extracted diffusion streamlines in 27 chronic schizophrenia and 26 healthy controls connecting 4 frontal subregions to the striatum. We labeled the projection zone striatal surface voxels into 2 subtypes: dominant-input from a single cortical subregion, and, functionally integrative, with mixed-input from diverse cortical subregions. We showed: 1) a group difference for total striatal surface voxel number (P = .045) driven by fewer mixed-input voxels in the left (P  = .007), but not right, hemisphere; 2) a group by hemisphere interaction for the ratio quotient between voxel subtypes (P  = .04) with a left (P  = .006), but not right, hemisphere increase in schizophrenia, also reflecting fewer mixed-input voxels; and 3) fewer mixed-input voxel counts in schizophrenia (P  = .045) driven by differences in left hemisphere limbic (P  = .007) and associative (P  = .01), but not sensorimotor, striatum. These results demonstrate a less integrative pattern of frontostriatal structural connectivity in chronic schizophrenia. A diminished integrative pattern yields a less complex input pattern to the striatum from the cortex with less circuit integration at the level of the striatum. Further, as brain wiring occurs during early development, aberrant brain wiring could serve as a developmental biomarker for schizophrenia.

Primary Cilia Signaling Promotes Axonal Tract Development and Is Disrupted in Joubert Syndrome-Related Disorders Models.

Appropriate axonal growth and connectivity are essential for functional wiring of the brain. Joubert syndrome-related disorders (JSRD), a group of ciliopathies in which mutations disrupt primary cilia function, are characterized by axonal tract malformations. However, little is known about how cilia-driven signaling regulates axonal growth and connectivity. We demonstrate that the deletion of related JSRD genes, Arl13b and Inpp5e, in projection neurons leads to de-fasciculated and misoriented axonal tracts. Arl13b deletion disrupts the function of its downstream effector, Inpp5e, and deregulates ciliary-PI3K/AKT signaling. Chemogenetic activation of ciliary GPCR signaling and cilia-specific optogenetic modulation of downstream second messenger cascades (PI3K, AKT, and AC3) commonly regulated by ciliary signaling receptors induce rapid changes in axonal dynamics. Further, Arl13b deletion leads to changes in transcriptional landscape associated with dysregulated PI3K/AKT signaling. These data suggest that ciliary signaling acts to modulate axonal connectivity and that impaired primary cilia signaling underlies axonal tract defects in JSRD.

The Evolution of Variability and Robustness in Neural Development.

As in all biological systems, neurons and their networks must balance precision with variability. Phenotypic precision and phenotypic variability can both occur with remarkable robustness, where robustness is defined as the ability to tolerate perturbation. Variability in genotype-phenotype mapping produces phenotypic variability despite identical genetic information. The resulting variability among genetically identical neurons can contribute to the robustness of brain development. Similarly, variability of genetically identical individuals can contribute to evolutionary robustness. We discuss here shared principles of developmental robustness and evolutionary robustness, and highlight scenarios where such principles result in neural networks that achieve robustness of precision or variability.

Seven pass Cadherins CELSR1-3.

Cadherin EGF LAG seven-pass G-type receptors 1, 2 and 3 (CELSR1-3) form a family of three atypical cadherins with multiple functions in epithelia and in the nervous system. During the past decade, evidence has accumulated for a key role of CELSR1 in epithelial planar cell polarity (PCP), and for CELSR2 and CELSR3 in ciliogenesis and neural development, especially neuron migration and axon guidance in the central, peripheral and enteric nervous systems. Phenotypes in mutant mice indicate that CELSR proteins work in concert with FZD3 and FZD6, but several questions remain. Apart from PCP signaling pathways implicating CELSR1 that begin to be unraveled, little is known about other signals generated by CELSR2 and CELSR3. A crucial question concerns the putative ligands that trigger signaling, in particular what is the role of WNT factors. Another critical issue is the identification of novel intracellular pathways and effectors that relay and transmit signals in receptive cells? Answers to those questions should further our understanding of the role of those important molecules not only in development but also in regeneration and disease.

miR-182 Regulates Slit2-Mediated Axon Guidance by Modulating the Local Translation of a Specific mRNA.

During brain wiring, cue-induced axon behaviors such as directional steering and branching are aided by localized mRNA translation. Different guidance cues elicit translation of subsets of mRNAs that differentially regulate the cytoskeleton, yet little is understood about how specific mRNAs are selected for translation. MicroRNAs (miRNAs) are critical translational regulators that act through a sequence-specific mechanism. Here, we investigate the local role of miRNAs in mRNA-specific translation during pathfinding of Xenopus laevis retinal ganglion cell (RGC) axons. Among a rich repertoire of axonal miRNAs, miR-182 is identified as the most abundant. Loss of miR-182 causes RGC axon targeting defects in vivo and impairs Slit2-induced growth cone (GC) repulsion. We find that miR-182 targets cofilin-1 mRNA, silencing its translation, and Slit2 rapidly relieves the repression without causing miR-182 degradation. Our data support a model whereby miR-182 reversibly gates the selection of transcripts for fast translation depending on the extrinsic cue.

The role of cell adhesion molecules in brain wiring and neuropsychiatric disorders.

Cell adhesion molecules (CAMs) in the nervous system have long been a research focus, but many mice lacking CAMs show very subtle phenotypes, giving an impression that CAMs may not be major players in constructing the nervous system. However, recent human genetic studies suggest CAM involvement in many neuropsychiatric disorders, implicating that they must have significant functions in nervous system development, namely in circuitry formation. As CAMs can provide specificity through their molecular interactions, this review summarizes possible mechanisms on how alterations of CAMs can result in neuropsychiatric disorders through circuitry modification.

Regulation of Drosophila Brain Wiring by Neuropil Interactions via a Slit-Robo-RPTP Signaling Complex.

The axonal wiring molecule Slit and its Round-About (Robo) receptors are conserved regulators of nerve cord patterning. Robo receptors also contribute to wiring brain circuits. Whether molecular mechanisms regulating these signals are modified to fit more complex brain wiring processes is unclear. We investigated the role of Slit and Robo receptors in wiring Drosophila higher-order brain circuits and identified differences in the cellular and molecular mechanisms of Robo/Slit function. First, we find that signaling by Robo receptors in the brain is regulated by the Receptor Protein Tyrosine Phosphatase RPTP69d. RPTP69d increases membrane availability of Robo3 without affecting its phosphorylation state. Second, we detect no midline localization of Slit during brain development. Instead, Slit is enriched in the mushroom body, a neuronal structure covering large areas of the brain. Thus, a divergent molecular mechanism regulates neuronal circuit wiring in the Drosophila brain, partly in response to signals from the mushroom body.

The evolution and development of neural superposition.

Visual systems have a rich history as model systems for the discovery and understanding of basic principles underlying neuronal connectivity. The compound eyes of insects consist of up to thousands of small unit eyes that are connected by photoreceptor axons to set up a visual map in the brain. The photoreceptor axon terminals thereby represent neighboring points seen in the environment in neighboring synaptic units in the brain. Neural superposition is a special case of such a wiring principle, where photoreceptors from different unit eyes that receive the same input converge upon the same synaptic units in the brain. This wiring principle is remarkable, because each photoreceptor in a single unit eye receives different input and each individual axon, among thousands others in the brain, must be sorted together with those few axons that have the same input. Key aspects of neural superposition have been described as early as 1907. Since then neuroscientists, evolutionary and developmental biologists have been fascinated by how such a complicated wiring principle could evolve, how it is genetically encoded, and how it is developmentally realized. In this review article, we will discuss current ideas about the evolutionary origin and developmental program of neural superposition. Our goal is to identify in what way the special case of neural superposition can help us answer more general questions about the evolution and development of genetically "hard-wired" synaptic connectivity in the brain.