Roles of odorant receptors during olfactory glomerular map formation.

The organization of the olfactory glomerular map involves the convergence of olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) into glomeruli in the olfactory bulb (OB). A remarkable feature of the olfactory glomerular map formation is that the identity of OR instructs the topography of the bulb, resulting in thousands of discrete glomeruli in mice. Several lines of evidence indicate that ORs control the expression levels of various kinds of transmembrane proteins to form glomeruli at appropriate regions of the OB. In this review, we will discuss how the OR identity is decoded by OSNs into gene expression through intracellular regulatory mechanisms.

Layer-Specific Inhibitory Microcircuits of Layer 6 Interneurons in Rat Prefrontal Cortex.

GABAergic interneurons in different cortical areas play important roles in diverse higher-order cognitive functions. The heterogeneity of interneurons is well characterized in different sensory cortices, in particular in primary somatosensory and visual cortex. However, the structural and functional properties of the medial prefrontal cortex (mPFC) interneurons have received less attention. In this study, a cluster analysis based on axonal projection patterns revealed four distinct clusters of L6 interneurons in rat mPFC: Cluster 1 interneurons showed axonal projections similar to Martinotti-like cells extending to layer 1, cluster 2 displayed translaminar projections mostly to layer 5, and cluster 3 interneuron axons were confined to the layer 6, whereas those of cluster 4 interneurons extend also into the white matter. Correlations were found between neuron location and axonal distribution in all clusters. Moreover, all cluster 1 L6 interneurons showed a monotonically adapting firing pattern with an initial high-frequency burst. All cluster 2 interneurons were fast-spiking, while neurons in cluster 3 and 4 showed heterogeneous firing patterns. Our data suggest that L6 interneurons that have distinct morphological and physiological characteristics are likely to innervate different targets in mPFC and thus play differential roles in the L6 microcircuitry and in mPFC-associated functions.

Early Forebrain Neurons and Scaffold Fibers in Human Embryos.

Neural progenitor proliferation, neuronal migration, areal organization, and pioneer axon wiring are critical events during early forebrain development, yet remain incompletely understood, especially in human. Here, we studied forebrain development in human embryos aged 5 to 8 postconceptional weeks (WPC5-8), stages that correspond to the neuroepithelium/early marginal zone (WPC5), telencephalic preplate (WPC6 & 7), and incipient cortical plate (WPC8). We show that early telencephalic neurons are formed at the neuroepithelial stage; the most precocious ones originate from local telencephalic neuroepithelium and possibly from the olfactory placode. At the preplate stage, forebrain organization is quite similar in human and mouse in terms of areal organization and of differentiation of Cajal-Retzius cells, pioneer neurons, and axons. Like in mice, axons from pioneer neurons in prethalamus, ventral telencephalon, and cortical preplate cross the diencephalon-telencephalon junction and the pallial-subpallial boundary, forming scaffolds that could guide thalamic and cortical axons at later stages. In accord with this model, at the early cortical plate stage, corticofugal axons run in ventral telencephalon in close contact with scaffold neurons, which express CELSR3 and FZD3, two molecules that regulates formation of similar scaffolds in mice.

Divergent topographic projection of cerebral cortical areas to overlapping cerebellar lobules through distinct regions of the pontine nuclei.

The massive axonal projection from the cerebrum to the cerebellum through the pontine nuclei supports the cerebrocerebellar coordination of motor and nonmotor functions. However, the cerebrum and cerebellum have distinct patterns of functional localization in their cortices. We addressed this issue by bidirectional neuronal tracing from 22 various locations of the pontine nuclei in the mouse in a comprehensive manner. Cluster analyses of the distribution patterns of labeled cortical pyramidal cells and cerebellar mossy fiber terminals classified all cases into six groups located in six different subareas of the pontine nuclei. The lateral (insular), mediorostral (cingulate and prefrontal), and caudal (visual and auditory) cortical areas of the cerebrum projected to the medial, rostral, and lateral subareas of the pontine nuclei, respectively. These pontine subareas then projected mainly to the crus I, central vermis, and paraflocculus divergently. The central (motor and somatosensory) cortical areas projected to the centrorostral, centrocaudal and caudal subareas of the pontine nuclei, which then projected mainly to the rostral and caudal lobules with a somatotopic arrangement. The results indicate a new pontine nuclei-centric view of the corticopontocerebellar projection: the generally parallel corticopontine projection to pontine nuclei subareas is relayed to the highly divergent pontocerebellar projection terminating in overlapping specific lobules of the cerebellum. Consequently, the mode of the pontine nuclei relay underlies the cerebellar functional organization.

Uncovering Statistical Links Between Gene Expression and Structural Connectivity Patterns in the Mouse Brain.

Finding links between genes and structural connectivity is of the utmost importance for unravelling the underlying mechanism of the brain connectome. In this study we identify links between the gene expression and the axonal projection density in the mouse brain, by applying a modified version of the Linked ICA method to volumetric data from the Allen Institute for Brain Science for identifying independent sources of information that link both modalities at the voxel level. We performed separate analyses on sets of projections from the visual cortex, the caudoputamen and the midbrain reticular nucleus, and we determined those brain areas, injections and genes that were most involved in independent components that link both gene expression and projection density data, while we validated their biological context through enrichment analysis. We identified representative and literature-validated cortico-midbrain and cortico-striatal projections, whose gene subsets were enriched with annotations for neuronal and synaptic function and related developmental and metabolic processes. The results were highly reproducible when including all available projections, as well as consistent with factorisations obtained using the Dictionary Learning and Sparse Coding technique. Hence, Linked ICA yielded reproducible independent components that were preserved under increasing data variance. Taken together, we have developed and validated a novel paradigm for linking gene expression and structural projection patterns in the mouse mesoconnectome, which can power future studies aiming to relate genes to brain function.

Topographically Distinct Projection Patterns of Early-Generated and Late-Generated Projection Neurons in the Mouse Olfactory Bulb.

In the mouse brain, olfactory information is transmitted to the olfactory cortex via olfactory bulb (OB) projection neurons known as mitral and tufted cells. Although mitral and tufted cells share many cellular characteristics, these cell types are distinct in their somata location and in their axonal and dendritic projection patterns. Moreover, mitral cells consist of heterogeneous subpopulations. We have previously shown that mitral cells generated at different embryonic days differentially localize within the mitral cell layer (MCL) and extend their lateral dendrites to different sublayers of the external plexiform layer (EPL). Here, we examined the axonal projection patterns from the subpopulations of OB projection neurons that are determined by the timing of neurogenesis (neuronal birthdate) to understand the developmental origin of the diversity in olfactory pathways. We separately labeled early-generated and late-generated OB projection neurons using in utero electroporation performed at embryonic day (E)11 and E12, respectively, and quantitatively analyzed their axonal projection patterns in the whole mouse brain using high-resolution 3D imaging. In this study, we demonstrate that the axonal projection of late-generated OB projection neurons is restricted to the anterior portion of the olfactory cortex while those of the early-generated OB projection neurons innervate the entire olfactory cortex. Our results suggest that the late-generated mitral cells do not extend their axons to the posterior regions of the olfactory cortex. Therefore, the mitral cells having different birthdates differ, not only in cell body location and dendritic projections within the OB, but also in their axonal projection pattern to the olfactory cortex.

Challenges in Modeling Human Neural Circuit Formation via Brain Organoid Technology.

Human brain organoids are three-dimensional self-organizing tissues induced from pluripotent cells that recapitulate some aspects of early development and some of the early structure of the human brain in vitro. Brain organoids consist of neural lineage cells, such as neural stem/precursor cells, neurons, astrocytes and oligodendrocytes. Additionally, brain organoids contain fluid-filled ventricle-like structures surrounded by a ventricular/subventricular (VZ/SVZ) zone-like layer of neural stem cells (NSCs). These NSCs give rise to neurons, which form multiple outer layers. Since these structures resemble some aspects of structural arrangements in the developing human brain, organoid technology has attracted great interest in the research fields of human brain development and disease modeling. Developmental brain disorders have been intensely studied through the use of human brain organoids. Relatively early steps in human brain development, such as differentiation and migration, have also been studied. However, research on neural circuit formation with brain organoids has just recently began. In this review, we summarize the current challenges in studying neural circuit formation with organoids and discuss future perspectives.

Prediction of a Cell-Class-Specific Mouse Mesoconnectome Using Gene Expression Data.

Reconstructing brain connectivity at sufficient resolution for computational models designed to study the biophysical mechanisms underlying cognitive processes is extremely challenging. For such a purpose, a mesoconnectome that includes laminar and cell-class specificity would be a major step forward. We analyzed the ability of gene expression patterns to predict cell-class and layer-specific projection patterns and assessed the functional annotations of the most predictive groups of genes. To achieve our goal we used publicly available volumetric gene expression and connectivity data and we trained computational models to learn and predict cell-class and layer-specific axonal projections using gene expression data. Predictions were done in two ways, namely predicting projection strengths using the expression of individual genes and using the co-expression of genes organized in spatial modules, as well as predicting binary forms of projection. For predicting the strength of projections, we found that ridge (L2-regularized) regression had the highest cross-validated accuracy with a median r2 score of 0.54 which corresponded for binarized predictions to a median area under the ROC value of 0.89. Next, we identified 200 spatial gene modules using a dictionary learning and sparse coding approach. We found that these modules yielded predictions of comparable accuracy, with a median r2 score of 0.51. Finally, a gene ontology enrichment analysis of the most predictive gene groups resulted in significant annotations related to postsynaptic function. Taken together, we have demonstrated a prediction workflow that can be used to perform multimodal data integration to improve the accuracy of the predicted mesoconnectome and support other neuroscience use cases.

Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex.

Astrocytes are particularly promising candidates for reprogramming into neurons, as they maintain some of the original patterning information from their radial glial ancestors. However, to which extent the position of astrocytes influences the fate of reprogrammed neurons remains unknown. To elucidate this, we performed stab wound injury covering an entire neocortical column, including the gray matter (GM) and white matter (WM), and targeted local reactive astrocytes via injecting FLEx switch (Cre-On) adeno-associated viral (AAV) vectors into mGFAP-Cre mice. Single proneural factors were not sufficient for adequate reprogramming, although their combination with the nuclear receptor-related 1 protein (Nurr1) improved reprogramming efficiency. Nurr1 and Neurogenin 2 (Ngn2) resulted in high-efficiency reprogramming of targeted astrocytes into neurons that develop lamina-specific hallmarks, including the appropriate long-distance axonal projections. Surprisingly, in the WM, we did not observe any reprogrammed neurons, thereby unveiling a crucial role of region- and layer-specific differences in astrocyte reprogramming.

Inhibitory interneurons and their circuit motifs in the many layers of the barrel cortex.

Recent years have seen substantial progress in studying the structural and functional properties of GABAergic interneurons and their roles in the neuronal networks of barrel cortex. Although GABAergic interneurons represent only about 12% of the total number of neocortical neurons, they are extremely diverse with respect to their structural and functional properties. It has become clear that barrel cortex interneurons not only serve the maintenance of an appropriate excitation/inhibition balance but also are directly involved in sensory processing. In this review we present different interneuron types and their axonal projection pattern framework in the context of the laminar and columnar organization of the barrel cortex. The main focus is here on the most prominent interneuron types, i.e. basket cells, chandelier cells, Martinotti cells, bipolar/bitufted cells and neurogliaform cells, but interneurons with more unusual axonal domains will also be mentioned. We describe their developmental origin, their classification with respect to molecular, morphological and intrinsic membrane and synaptic properties. Most importantly, we will highlight the most prominent circuit motifs these interneurons are involved in and in which way they serve feed-forward inhibition, feedback inhibition and disinhibition. Finally, this will be put into context to their functional roles in sensory signal perception and processing in the whisker system and beyond.

Neurons Generated by Mouse ESCs with Hippocampal or Cortical Identity Display Distinct Projection Patterns When Co-transplanted in the Adult Brain.

The capability of generating neural precursor cells with distinct types of regional identity in vitro has recently opened new opportunities for cell replacement in animal models of neurodegenerative diseases. By manipulating Wnt and BMP signaling, we steered the differentiation of mouse embryonic stem cells (ESCs) toward isocortical or hippocampal molecular identity. These two types of cells showed different degrees of axonal outgrowth and targeted different regions when co-transplanted in healthy or lesioned isocortex or in hippocampus. In hippocampus, only precursor cells with hippocampal molecular identity were able to extend projections, contacting CA3. Conversely, isocortical-like cells were capable of extending long-range axonal projections only when transplanted in motor cortex, sending fibers toward both intra- and extra-cortical targets. Ischemic damage induced by photothrombosis greatly enhanced the capability of isocortical-like cells to extend far-reaching projections. Our results indicate that neural precursors generated by ESCs carry intrinsic signals specifying axonal extension in different environments.

A Visual-Cue-Dependent Memory Circuit for Place Navigation.

The ability to remember and to navigate to safe places is necessary for survival. Place navigation is known to involve medial entorhinal cortex (MEC)-hippocampal connections. However, learning-dependent changes in neuronal activity in the distinct circuits remain unknown. Here, by using optic fiber photometry in freely behaving mice, we discovered the experience-dependent induction of a persistent-task-associated (PTA) activity. This PTA activity critically depends on learned visual cues and builds up selectively in the MEC layer II-dentate gyrus, but not in the MEC layer III-CA1 pathway, and its optogenetic suppression disrupts navigation to the target location. The findings suggest that the visual system, the MEC layer II, and the dentate gyrus are essential hubs of a memory circuit for visually guided navigation.

Effect of draxin knockdown on the projection of 23C10-positive neural fibers in the embryonic chick hindbrain / 解剖学报

[Abstract] Objective To investigate the effects of the downregulation of draxin expression on the projection characteristics of 23C10-positive neural fibers in the chick embryonic hindbrain. Methods The vitro incubation of HH stages 21-22 chick embryonic hindbrain biopsy with alkaline phosphatase (ALP) protein was used as control group. The incubation of HH stages 21-22 chick embryonic hindbrain biopsy with draxin-ALP fusion protein was used as experimental group. The number of embryonic hindbrain for each group was 10. To detect whether 23C10-positive neural fibers could directly bind to draxin protein or not;In ovo electroporation using empty vector in the chick embryonic hindbrain was used as control group. In ovo electroporation with small interfering RNA(siRNA) expressing vector for reducing draxin expression in the chick embryonic hindbrain was used as experimental group. The number of embryonic hindbrain for each group was 18. The effect of the down-regulation of draxin expression and the change of projection characteristics of 23C10-positive neural fibers were observed to check whether the down-regulation of draxin expression would affect the distribution of 23C10-positive fibers. Results Most portion of draxin protein could overlap with 23C10-positive neural fibers in HH stages 21-22 chick embryonic hindbrain biopsies; After expression of the siRNA plasmid against draxin by electroporation, the expression level of draxin protein was significantly reduced, and the distribution of 23C10-positive fibers was scattered in the dorsal hindbrain on the electroporated side at HH stages 25-26 of chick embryos (P < 0. 05) . Conclusion Draxin protein may directly bind to 23C10-positive fibers in hindbrain, and it plays an important regulatory role in the fasciculation of 23C10-positive fibers during chick embryonic development.

Contribution of the Runx1 transcription factor to axonal pathfinding and muscle innervation by hypoglossal motoneurons.

The runt-related transcription factor Runx1 contributes to cell type specification and axonal targeting projections of the nociceptive dorsal root ganglion neurons. Runx1 is also expressed in the central nervous system, but little is known of its functions in brain development. At mouse embryonic day (E) 17.5, Runx1-positive neurons were detected in the ventrocaudal subdivision of the hypoglossal nucleus. Runx1-positive neurons lacked calcitonin gene-related peptide (CGRP) expression, whereas Runx1-negative neurons expressed CGRP. Expression of CGRP was not changed in Runx1-deficient mice at E17.5, suggesting that Runx1 alone does not suppress CGRP expression. Hypoglossal axon projections to the intrinsic vertical (V) and transverse (T) tongue muscles were sparser in Runx1-deficient mice at E17.5 compared to age-matched wild-type littermates. Concomitantly, vesicular acetylcholine transporter-positive axon terminals and acetylcholine receptor clusters were less dense in the V and T tongue muscles of Runx1-deficient mice. These abnormalities in axonal projection were not caused by a reduction in the total number hypoglossal neurons, failed synaptogenesis, or tongue muscles deficits. Our results implicate Runx1 in the targeting of ventrocaudal hypoglossal axons to specific tongue muscles. However, Runx1 deficiency did not alter neuronal survival or the expression of multiple motoneuron markers as in other neuronal populations. Thus, Runx1 appears to have distinct developmental functions in different brain regions.

Brain-specific transcriptional regulator T-brain-1 controls brain wiring and neuronal activity in autism spectrum disorders.

T-brain-1 (TBR1) is a brain-specific T-box transcription factor. In 1995, Tbr1 was first identified from a subtractive hybridization that compared mouse embryonic and adult telencephalons. Previous studies of Tbr1 (-∕-) mice have indicated critical roles for TBR1 in the development of the cerebral cortex, amygdala, and olfactory bulb. Neuronal migration and axonal projection are two important developmental features controlled by TBR1. Recently, recurrent de novo disruptive mutations in the TBR1 gene have been found in patients with autism spectrum disorders (ASDs). Human genetic studies have identified TBR1 as a high-confidence risk factor for ASDs. Because only one allele of the TBR1 gene is mutated in these patients, Tbr1 (+∕-) mice serve as a good genetic mouse model to explore the mechanism by which de novo TBR1 mutation leads to ASDs. Although neuronal migration and axonal projection defects of cerebral cortex are the most prominent phenotypes in Tbr1 (-∕-) mice, these features are not found in Tbr1 (+∕-) mice. Instead, inter- and intra-amygdalar axonal projections and NMDAR expression and activity in amygdala are particularly susceptible to Tbr1 haploinsufficiency. The studies indicated that both abnormal brain wiring (abnormal amygdalar connections) and excitation/inhibition imbalance (NMDAR hypoactivity), two prominent models for ASD etiology, are present in Tbr1 (+∕-) mice. Moreover, calcium/calmodulin-dependent serine protein kinase (CASK) was found to interact with TBR1. The CASK-TBR1 complex had been shown to directly bind the promoter of the Grin2b gene, which is also known as Nmdar2b, and upregulate Grin2b expression. This molecular function of TBR1 provides an explanation for NMDAR hypoactivity in Tbr1 (+∕-) mice. In addition to Grin2b, cell adhesion molecules-including Ntng1, Cdh8, and Cntn2-are also regulated by TBR1 to control axonal projections of amygdala. Taken together, the studies of Tbr1 provide an integrated picture of ASD etiology at the cellular and circuit levels.

Developmental regulation of neural map formation in the mouse olfactory system.

In the mouse olfactory system, various odorants are detected by approximately 1000 different odorant receptors (ORs) expressed in the olfactory sensory neurons (OSNs). It is well established that each OSN expresses only one functional OR gene in a monoallelic manner. Furthermore, OSN axons expressing the same OR converge to a set of glomeruli in the olfactory bulb (OB). During embryonic development, a coarse map is formed by the combination of two genetically programmed processes. One is OR-independent axonal projection along the dorsal-ventral (D-V) axis, and the other is OR-dependent projection along the anterior-posterior (A-P) axis. D-V projection is regulated by the anatomical location of OSNs within the olfactory epithelium (OE), whereas A-P projection is instructed by expressed OR molecules using cyclic adenosine monophosphate (cAMP) signals. After birth, the map is further refined in an activity-dependent manner by its conversion from a continuous to a discrete map through segregation of glomerular structures. Here, we summarize recent progress from our laboratory in understanding neural map formation in the mouse olfactory system.

Three axonal projection routes of individual pyramidal cells in the ventral CA1 hippocampus.

Pyramidal cells of the ventral hippocampal CA1 area have numerous and diverse distant projections to other brain regions including the temporal and parietal association areas, visual, auditory, olfactory, somatosensory, gustatory, and visceral areas, and inputs to the amygdalar and prefrontal-orbital-agranular insular region. In addition, their differential expression of proteins like calbindin provides further indications for cellular diversity. This raises the possibility that the pyramidal cells may form subpopulations participating in different brain circuitries. To address this hypothesis we applied the juxtacellular labeling technique to fill individual pyramidal cells in the ventral hippocampus with neurobiotin in urethane anesthetized rats. For each labeled pyramidal cell we determined soma location, dendritic arborizations and selective expression of calbindin and norbin. Reconstruction and mapping of long-range axonal projections were made with the Neurolucida system. We found three major routes of ventral CA1 pyramidal cell projections. The classical pathway run caudo-ventrally across and innervating the subiculum, further to the parahippocampal regions and then to the deep and superficial layers of entorhinal cortex. The other two pathways avoided subiculum by branching from the main axon close to the soma and either traveled antero- and caudo-ventrally to amygdaloid complex, amygdalopiriform-transition area and parahippocampal regions or run antero-dorsally through the fimbria-fornix to the septum, hypothalamus, ventral striatum and olfactory regions. We found that most pyramidal cells investigated used all three major routes to send projecting axons to other brain areas. Our results suggest that the information flow through the ventral hippocampus is distributed by wide axonal projections from the CA1 area.

Morphological characterization of respiratory neurons in the pre-Bötzinger complex.

Although the pre-Bötzinger complex (preBötC) was defined as the inspiratory rhythm generator long ago, the functional-anatomical characterization of its neuronal components is still being achieved. Recent advances have identified the expression of molecular markers in the preBötC neurons that, however, are not exclusive to specific respiratory neuron subtypes and have not always been related to specific cell morphologies. Here, we evaluated the morphology and the axonal projections of electrophysiologically defined respiratory neurons in the preBötC using whole-cell recordings and intracellular biocytin labeling. We found that respiratory pacemaker neurons are larger than expiratory neurons and that inspiratory neurons are smaller than pacemaker and expiratory neurons. Other morphological features such as somata shapes or dendritic branching patterns were not found to be significantly different among the preBötC neurons sampled. We also found that both pacemaker and inspiratory nonpacemaker neurons, but not expiratory neurons, show extensive axonal projections to the contralateral preBötC and show signs of electrical coupling. Overall, our data suggest that there are morphological differences between subtypes of preBötC respiratory neurons. It will be important to take such differences in consideration since morphological differences would influence synaptic responses and action potential propagation.

Possible roles of Robo1+ ensheathing cells in guiding dorsal-zone olfactory sensory neurons in mouse.

In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) correlate with their axonal projection sites along the dorsoventral axis of the olfactory bulb (OB). We have previously reported that Neuropilin-2 expressed by ventral-zone OSNs contributes to the segregation of dorsal and ventral OSN axons, and that Slit is acting as a negative land mark to restrict the projection of Robo2+, early-arriving OSN axons to the embryonic OB. Here, we report that another guidance receptor, Robo1, also plays an important role in guiding OSN axons. Knockout mice for Robo1 demonstrated defects in targeting of OSN axons to the OB. Although Robo1 is colocalized with dorsal-zone OSN axons, it is not produced by OSNs, but instead by olfactory ensheathing cells. These findings indicate a novel strategy of axon guidance in the mouse olfactory system during development.