Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism.

Substantial evidence suggests that chromosomal abnormalities contribute to the risk of autism. The duplication of human chromosome 15q11-13 is known to be the most frequent cytogenetic abnormality in autism. We have modeled this genetic change in mice by using chromosome engineering to generate a 6.3 Mb duplication of the conserved linkage group on mouse chromosome 7. Mice with a paternal duplication display poor social interaction, behavioral inflexibility, abnormal ultrasonic vocalizations, and correlates of anxiety. An increased MBII52 snoRNA within the duplicated region, affecting the serotonin 2c receptor (5-HT2cR), correlates with altered intracellular Ca(2+) responses elicited by a 5-HT2cR agonist in neurons of mice with a paternal duplication. This chromosome-engineered mouse model for autism seems to replicate various aspects of human autistic phenotypes and validates the relevance of the human chromosome abnormality. This model will facilitate forward genetics of developmental brain disorders and serve as an invaluable tool for therapeutic development.

Evidence That the Laminar Fate of LGE/CGE-Derived Neocortical Interneurons Is Dependent on Their Progenitor Domains.

Neocortical interneurons show tremendous diversity in terms of their neurochemical marker expressions, morphology, electrophysiological properties, and laminar fate. Allocation of interneurons to their appropriate regions and layers in the neocortex is thought to play important roles for the emergence of higher functions of the neocortex. Neocortical interneurons mainly originate from the medial ganglionic eminence (MGE) and the caudal ganglionic eminence (CGE). The diversity and the laminar fate of MGE-derived interneurons depend on the location of their birth and birthdate, respectively. However, this relationship does not hold for CGE-derived interneurons. Here, using the method of in utero electroporation, which causes arbitrary occurrence of labeled progenitor domains, we tracked all descendants of the lateral ganglionic eminence (LGE)/CGE progenitors in mice. We provide evidence that neocortical interneurons with distinct laminar fate originate from distinct progenitor domains within the LGE/CGE. We find layer I interneurons are predominantly labeled in a set of animals, whereas other upper layer neurons are predominantly labeled in another set. We also find distinct subcortical structures labeled between the two sets. Further, interneurons labeled in layer I show distinct neurochemical properties from those in other layers. Together, these results suggest that the laminar fate of LGE/CGE-derived interneurons depends on their spatial origin. SIGNIFICANCE STATEMENT: Diverse types of neocortical interneurons have distinct laminar fate, neurochemical marker expression, morphology, and electrophysiological properties. Although the specifications and laminar fate of medial ganglionic eminence-derived neocortical interneurons depend on their location of embryonic origin and birthdate, no similar causality of lateral/caudal ganglionic eminence (LGE/CGE)-derived neocortical interneurons is known. Here, we performed in utero electroporation on mouse LGE/CGE and found two groups of animals, one with preferential labeling of layer I and the other with preferential labeling of other layers. Interneurons labeled in these two groups show distinct neurochemical properties and morphologies and are associated with labeling of distinct subcortical structures. These findings suggest that the laminar fate of LGE/CGE-derived neocortical interneurons depends on their spatial origin.

Cortical Divergent Projections in Mice Originate from Two Sequentially Generated, Distinct Populations of Excitatory Cortical Neurons with Different Initial Axonal Outgrowth Characteristics.

Excitatory cortical neurons project to various subcortical and intracortical regions, and exhibit diversity in their axonal connections. Although this diversity may develop from primary axons, how many types of axons initially occur remains unknown. Using a sparse-labeling in utero electroporation method, we investigated the axonal outgrowth of these neurons in mice and correlated the data with axonal projections in adults. Examination of lateral cortex neurons labeled during the main period of cortical neurogenesis (E11.5-E15.5) indicated that axonal outgrowth commonly occurs in the intermediate zone. Conversely, the axonal direction varied; neurons labeled before E12.5 and the earliest cortical plate neurons labeled at E12.5 projected laterally, whereas neurons labeled thereafter projected medially. The expression of Ctip2 and Satb2 and the layer destinations of these neurons support the view that lateral and medial projection neurons are groups of prospective subcortical and callosal projection neurons, respectively. Consistently, birthdating experiments demonstrated that presumptive lateral projection neurons were generated earlier than medial projection neurons, even within the same layer. These results suggest that the divergent axonal connections of excitatory cortical neurons begin from two types of primary axons, which originate from two sequentially generated distinct subpopulations: early-born lateral (subcortical) and later-born medial (callosal) projection neuron groups.

Excitatory cortical neurons with multipolar shape establish neuronal polarity by forming a tangentially oriented axon in the intermediate zone.

The formation of axon-dendrite polarity is crucial for neuron to make the proper information flow within the brain. Although the processes of neuronal polarity formation have been extensively studied using neurons in dissociated culture, the corresponding developmental processes in vivo are still unclear. Here, we illuminate the initial steps of morphological polarization of excitatory cortical neurons in situ, by sparsely labeling their neuroepithelial progenitors using in utero electroporation and then examining their neuronal progeny in brain sections and in slice cultures. Morphological analysis showed that an axon-like long tangential process formed in progeny cells in the intermediate zone (IZ). Time-lapse imaging analysis using slice culture revealed that progeny cells with multipolar shape, after alternately extending and retracting their short processes for several hours, suddenly elongated a long process tangentially. These cells then transformed into a bipolar shape, extending a pia-directed leading process, and migrated radially leaving the tangential process behind, which gave rise to an "L-shaped" axon. Our findings suggest that neuronal polarity in these cells is established de novo from a nonpolarized stage in vivo and indicate that excitatory cortical neurons with multipolar shape in the IZ initiate axon outgrowth before radial migration into the cortical plate.

Role of neuropilin-2 in the ipsilateral growth of midbrain dopaminergic axons.

Axonal projections in the CNS can be categorized as either crossed or uncrossed. Crossing and uncrossing of axons has been explained by attractive and repulsive molecules like Netrin-1 and Slits, which are secreted by midline structures. However, uncrossed projections can be established even in double knockout mice of slit1 and slit2 or of roundabout1 (robo1) and robo2, two receptors for Slits. Here, we found that a novel mechanism mediated by Neuropilin-2 (Nrp2) contributes to the formation of uncrossed projections of midbrain dopaminergic neurons (mDANs). Nrp2 transcriptional activities were detected in a subset of mDANs, and its protein was expressed in mDAN axons growing through the ipsilateral diencephalon. In nrp2(lac) (Z) (/lac) (Z) mice, mDAN axons aberrantly grew toward the ventral midline and even crossed it, suggesting that Nrp2 is necessary for the development of mDAN ipsilateral projections. We investigated the involvement of Semaphorin 3B (Sema3B) and Sema3F, two ligands of Nrp2, by analysing mDAN axon trajectories in single or double knockout mice. In both cases, mDAN axons still projected ipsilaterally, suggesting the involvement mechanisms independent of these Sema3s. Nrp2-deficient mDAN axons retained their responsiveness to Slit2, demonstrating that aberrant mDAN axons in nrp2(lac) (Z) (/lac) (Z) mice were not indirectly mediated by alterations in Slit/Robo signaling. Taken together, our results indicate that a novel mechanism mediated by Nrp2 contributes to the establishment of uncrossed projections by mDAN axons.

Specific AAV2/PHP.eB-mediated gene transduction of CA2 pyramidal cells via injection into the lateral ventricle.

Given its limited accessibility, the CA2 area has been less investigated compared to other subregions of the hippocampus. While the development of transgenic mice expressing Cre recombinase in the CA2 has revealed unique features of this area, the use of mouse lines has several limitations, such as lack of specificity. Therefore, a specific gene delivery system is required. Here, we confirmed that the AAV-PHP.eB capsid preferably infected CA2 pyramidal cells following retro-orbital injection and demonstrated that the specificity was substantially higher after injection into the lateral ventricle. In addition, a tropism for the CA2 area was observed in organotypic slice cultures. Combined injection into the lateral ventricle and stereotaxic injection into the CA2 area specifically introduced the transgene into CA2 pyramidal cells, enabling us to perform targeted patch-clamp recordings and optogenetic manipulation. These results suggest that AAV-PHP.eB is a versatile tool for specific gene transduction in CA2 pyramidal cells.

Preferential arborization of dendrites and axons of parvalbumin- and somatostatin-positive GABAergic neurons within subregions of the mouse claustrum.

The claustrum coordinates the activities of individual cortical areas through abundant reciprocal connections with the cerebral cortex. Although these excitatory connections have been extensively investigated in three subregions of the claustrum-core region and dorsal and ventral shell regions-the contribution of GABAergic neurons to the circuitry in each subregion remains unclear. Here, we examined the distribution of GABAergic neurons and their dendritic and axonal arborizations in each subregion. Combining in situ hybridization with immunofluorescence histochemistry showed that approximately 10% of neuronal nuclei-positive cells expressed glutamic acid decarboxylase 67 mRNA across the claustral subregions. Approximately 20%, 30%, and 10% of GABAergic neurons were immunoreactive for parvalbumin (PV), somatostatin (SOM), and vasoactive intestinal polypeptide, respectively, in each subregion, and these neurochemical markers showed little overlap with each other. We then reconstructed PV and SOM neurons labeled with adeno-associated virus vectors. The dendrites and axons of PV and SOM neurons were preferentially localized to their respective subregions where their cell bodies were located. Furthermore, the axons were preferentially extended in a rostrocaudal direction, whereas the dendrites were relatively isotropic. The present findings suggest that claustral PV and SOM neurons might execute information processing separately within the core and shell regions.

Formation of axon-dendrite polarity in situ: initiation of axons from polarized and non-polarized cells.

Neurons are polarized cells that extend a single axon and several dendrites. Historically, how neurons establish their axon-dendrite polarity has been extensively studied using dissociated hippocampal cells in culture. Although such studies have identified the cellular and molecular mechanisms underlying axon-dendrite polarization, the conclusions have been limited to in vitro conditions. Recent progress using live imaging has enabled us to directly observe axon formation in situ, revealing distinct cellular mechanisms that regulate axon-dendrite polarization in vivo. In this review, we compare the cellular events during axon formation studied in various systems both in vivo and in vitro and discuss possible common mechanisms underlying the axon-dendrite polarization.

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales.

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (-), and ScaleS4 solution, for a total of 10.5-14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

Protocol for multi-scale light microscopy/electron microscopy neuronal imaging in mouse brain tissue.

An imaging technique across multiple spatial scales is required for extracting structural information on neurons with processes of meter scale length and specialized nanoscale structures. Here, we present a protocol combining multi-scale light microscopy (LM) with electron microscopy (EM) in mouse brain tissue. We describe tissue slice preparation and LM/EM dual labeling with EGFP-APEX2 fusion protein. We then detail ScaleSF tissue clearing and successive LM/EM imaging. Our protocol allows for deciphering structural information across multiple spatial scales on neurons. For complete details on the use and execution of this protocol, please refer to Furuta et al. (2022).

Multi-scale light microscopy/electron microscopy neuronal imaging from brain to synapse with a tissue clearing method, ScaleSF.

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

Fluorochromized tyramide-glucose oxidase as a multiplex fluorescent tyramide signal amplification system for histochemical analysis.

Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, biotinyl tyramine-glucose oxidase (BT-GO), for bright-filed imaging. Here, we develop fluorochromized tyramide-glucose oxidase (FT-GO) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of fluorochromized tyramide (FT) with hydrogen peroxide produced by enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched antibody-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.

Application of a Tissue Clearing Method for the Analysis of Dopaminergic Axonal Projections.

Parkinson's disease (PD) is a neurodegenerative disorder characterized with the progressive loss of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNc). Quantitative analysis of neuronal loss including neuronal processes, axons and dendrites, would advance the understanding of the pathogenesis of PD. ScaleS, an aqueous tissue clearing method, provides stable tissue preservation while maintaining potent clearing capability, allowing quantitative three-dimensional (3D) imaging of biological tissues. In this chapter, we describe detailed procedures for 3D imaging of brain slice tissues with ScaleS technique. These include brain slice preparation, tissue clarification, chemical and immunohistochemical labeling (ChemScale and AbScale), and observation of labeled tissues using a confocal laser scanning microscope (CLSM).

Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system.

We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).

FGF8 signaling regulates growth of midbrain dopaminergic axons by inducing semaphorin 3F.

Accumulating evidence indicates that signaling centers controlling the dorsoventral (DV) polarization of the neural tube, the roof plate and the floor plate, play crucial roles in axon guidance along the DV axis. However, the role of signaling centers regulating the rostrocaudal (RC) polarization of the neural tube in axon guidance along the RC axis remains unknown. Here, we show that a signaling center located at the midbrain-hindbrain boundary (MHB) regulates the rostrally directed growth of axons from midbrain dopaminergic neurons (mDANs). We found that beads soaked with fibroblast growth factor 8 (FGF8), a signaling molecule that mediates patterning activities of the MHB, repelled mDAN axons that extended through the diencephalon. This repulsion may be mediated by semaphorin 3F (sema3F) because (1) FGF8-soaked beads induced an increase in expression of sema3F, (2) sema3F expression in the midbrain was essentially abolished by the application of an FGF receptor tyrosine kinase inhibitor, and (3) mDAN axonal growth was also inhibited by sema3F. Furthermore, mDAN axons expressed a sema3F receptor, neuropilin-2 (nrp2), and the removal of nrp-2 by gene targeting caused caudal growth of mDAN axons. These results indicate that the MHB signaling center regulates the growth polarity of mDAN axons along the RC axis by inducing sema3F.

Overlapping Projections of Neighboring Direct and Indirect Pathway Neostriatal Neurons to Globus Pallidus External Segment.

Indirect pathway medium-sized spiny neurons (iMSNs) in the neostriatum are well known to project to the external segment of the globus pallidus (GPe). Although direct MSNs (dMSNs) also send axon collaterals to the GPe, it remains unclear how dMSNs and iMSNs converge within the GPe. Here, we selectively labeled neighboring dMSNs and iMSNs with green and red fluorescent proteins using an adeno-associated virus vector and examined axonal projections of dMSNs and iMSNs to the GPe in mice. Both dMSNs and iMSNs formed two axonal arborizations displaying topographical projections in the dorsoventral and mediolateral planes. iMSNs displayed a wider and denser axon distribution, which included that of dMSNs. Density peaks of dMSN and iMSN axons almost overlapped, revealing convergence of dMSN axons in the center of iMSN projection fields. These overlapping projections suggest that dMSNs and iMSNs may work cooperatively via interactions within the GPe.

Netrin-1 Derived from the Ventricular Zone, but not the Floor Plate, Directs Hindbrain Commissural Axons to the Ventral Midline.

Netrin-1 (Ntn1) emanating from the ventral midline has been thought to act as a long-range diffusible chemoattractant for commissural axons (CAs). However, CAs still grow towards the midline in the absence of the floor plate (FP), a glial structure occupying the midline. Here, using genetically loss-of-function approaches in mice, we show that Ntn1 derived from the ventricular zone (VZ), but not the FP, is crucial for CA guidance in the mouse hindbrain. During the period of CA growth, Ntn1 is expressed in the ventral two-thirds of the VZ, in addition to the FP. Remarkably, deletion of Ntn1 from the VZ and even from the dorsal VZ highly disrupts CA guidance to the midline, whereas the deletion from the FP has little impact on it. We also show that the severities of CA guidance defects found in the Ntn1 conditional mutants were irrelevant to their FP long-range chemoattractive activities. Our results are incompatible with the prevailing view that Ntn1 is an FP-derived long-range diffusible chemoattractant for CAs, but suggest a novel mechanism that VZ-derived Ntn1 directs CAs to the ventral midline by its local actions.

Association of astrocytes with neurons and astrocytes derived from distinct progenitor domains in the subpallium.

Astrocytes play pivotal roles in metabolism and homeostasis as well as in neural development and function in a manner thought to depend on their region-specific diversity. In the mouse spinal cord, astrocytes and neurons, which are derived from a common progenitor domain (PD) and controlled by common PD-specific transcription factors, migrate radially and share their final positions. However, whether astrocytes can only interact with neurons from common PDs in the brain remains unknown. Here, we focused on subpallium-derived cells, because the subpallium generates neurons that show a diverse mode of migration. We tracked their fate by in utero electroporation of plasmids that allow for chromosomal integration of transgenes or of a Cre recombinase expression vector to reporter mice. We also used an Nkx2.1(Cre) mouse line to fate map the cells originating from the medial ganglionic eminence and preoptic area. We find that although neurons and astrocytes are labeled in various regions, only neurons are labeled in the neocortex, hippocampus and olfactory bulb. Furthermore, we find astrocytes derived from an Nkx 2.1-negative PD are associated with neurons from the Nkx2.1(+) PD. Thus, forebrain astrocytes can associate with neurons as well as astrocytes derived from a distinct PD.

Draft Genome Sequence of the Archiascomycetous Yeast Saitoella complicata.

The draft genome sequence of the archiasomycetous yeast Saitoella complicata was determined. The assembly of newly and previously sequenced data sets resulted in 104 contigs (total of 14.1 Mbp; N 50, 239 kbp). On the newly assembled genome, a total of 6,933 protein-coding sequences (7,119 transcripts, including alternative splicing forms) were identified.

Temporally- and spatially regulated generation of distinct descendants by sonic hedgehog-expressing progenitors in the forebrain.

The generation of distinct neural subtypes depends on the activities of cell-extrinsic and -intrinsic factors during the development of the vertebrate CNS. Previous studies have provided a molecular basis for how neural progenitors are patterned and generate distinct descendants that are spatially and temporally regulated by inductive signals secreted by polarized sources. However, it still remains unknown how the generation of neural descendants by progenitors located at polarized sources of inductive signals is controlled. Sonic hedgehog (Shh), which is expressed at the ventral midline in the forebrain, has been shown to play a critical role for the patterning and specification of distinct neural subtypes in the forebrain. Here, we analyzed the identities and distributions of Shh-descendants generated at discrete time points in the forebrain by using a ShhcreER(T2) mouse driver line in which a tamoxifen-inducible Cre cassette was inserted into the Shh locus together with a Z/EG mouse reporter line. Our results showed that Shh-expressing neural progenitors generated neuronal and glial descendants distributed throughout the telencephalon and diencephalon in a temporally distinct manner. Furthermore, our results showed that Shh-progenitors are located at two spatially distinct sub-domains that can be characterized by their temporally distinct patterns of Shh expression. These results suggest that temporally- and spatially controlled mechanisms that specify neural subtypes operate in the Shh-expressing progenitor domain, and raise the possibility that the distinct temporal gradient of Shh activity might be responsible for the generation of distinct neural subtypes in the telencephalon.