Early Postnatal Mouse Brain Research Articles

Endothelial Rbpj Is Required for Cerebellar Morphogenesis and Motor Control in the Early Postnatal Mouse Brain.

Intercellular influences are necessary for coordinated development and function of vascular and neural components in the brain. In the early postnatal period after birth, the mammalian cerebellum undergoes extensive morphogenesis - developing its characteristic lobules, organizing its diverse cell types into defined cellular layers, and establishing neural circuits that support cerebellar function, such as coordinated movement. In parallel, the cerebellar vasculature undergoes extensive postnatal growth and maturation, keeping pace with the expanding neural compartment. Endothelial deletion of Rbpj leads to neurovascular abnormalities in mice, including arteriovenous (AV) shunts that supplant capillaries and instead direct high-pressure/high-flow arterial blood directly to veins. Gross and histopathological cerebellar abnormalities, associated with these Rbpj-mediated brain AV malformations (AVMs), led to our hypothesis that early postnatal morphogenesis and lamination of cerebellum was perturbed in mice harboring endothelial Rbpj deficiency from birth. Here, we show that endothelial Rbpj-mutant mice developed enlarged vascular malformations on the cerebellar surface, by 2-week post-Rbpj deletion. In addition, outgrowth of cerebellar lobules was impaired through decreased cell proliferation, but not increased apoptosis, in the external granule layer. Molecular layer thickness was reduced, and the Purkinje layer was affected, by decreased Purkinje cell number, primary dendrite length, and dendritic arbor density. Endothelial deletion of Rbpj also led to impaired motor behaviors, consistent with abnormal cerebellar morphogenesis and lamination. Thus, our data suggest that Rbpj is required, in early postnatal vascular endothelium, to ensure proper cerebellar outgrowth, morphogenesis, and function in mice.

Parallel functional testing identifies enhancers active in early postnatal mouse brain.

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type-specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

NGPF2 triggers synaptic scaling up through ALK-LIMK-cofilin-mediated mechanisms.

Synaptic scaling is an extensively studied form of homeostatic plasticity critically involved in various brain functions. Although it is accepted that synaptic scaling is expressed through the postsynaptic accumulation of AMPA receptors (AMPARs), the induction mechanism remains elusive. In this study, we show that TTX treatment induces rapid but transient release of the neurite growth-promoting factor 2 (NGPF2), and this release is necessary and sufficient for TTX-induced scaling up. In addition, we show that inhibition of the anaplastic lymphoma kinase (ALK)-LIMK-cofilin signaling pathway blocks TTX- and NGPF2-induced synaptic scaling up. Furthermore, we show that TTX-induced release of NGPF2 is protein synthesis dependent and requires fragile X mental retardation protein 1 (FMRP1). These results indicate that activity blockade induces NGPF2 synthesis and release to trigger synaptic scaling up through LIMK-cofilin-dependent actin reorganization, spine enlargement, and stabilization of AMPARs at the synapse.

Optimized Immunostaining of Embryonic and Early Postnatal Mouse Brain Sections.

The mammalian neocortex, the outer layer of the cerebrum and most recently evolved brain region, is characterized by its unique areal and laminar organization. Distinct cortical layers and areas can be identified by the protein expression of graded transcription factors and molecular determinants that define the identity of different projection neurons. Thus, specific detection and visualization of protein expression is crucial for assessing the identity of neocortical neurons and, more broadly, for understanding early and late developmental mechanisms and function of this complex system. Several immunostaining/immunofluorescence methods exist to detect protein expression. Published protocols vary with regard to subtle details, which may impact the final outcome of the immunofluorescence. Here, we provide a detailed protocol, suitable for both thin cryostat sections and thick vibratome sections, which has successfully worked for a wide range of antibodies directed against key molecular players of neocortical development. Ranging from early technical steps of brains collection down to image analysis and statistics, we include every detail concerning sample inclusion and sectioning, slide storage and optimal antibody dilutions aimed at reducing non-specific background. Routinely used in the lab, our background-optimized immunostaining protocol allows efficient detection of area- and layer- specific molecular determinants of distinct neocortical projection neurons. Graphic abstract: Workflow chart for the optimized immunostaining protocol of mouse brain sections. A. A flow chart for different steps of the optimized immunostaining protocol on both thin cryostat and thick vibratome sections. B. Example for immunostaining against Satb2 and Ctip2 on a thin coronal section (20 μm) at the level of the somatosensory cortex. The first column to the left shows the binning system where 6 bins can be overlaid on the image. On the bottom, an example of counting analysis showing the percentage of marker-positive cells normalized to the total number of DAPI or Hoechst-positive cells. C. Example for immunostaining against Satb2 and Ctip2 on a GFP+ thick vibratome section (200 μm). Images are taken at low magnification (10x, left) and high magnification (40x, right). The graph shows a counting of the percentage of Ctip2-positive neurons normalized to the total number of GFP-electroporated neurons on high-magnification images. Images on B and C are modified from Harb et al. (2016).

Rapastinel alleviates the neurotoxic effect induced by NMDA receptor blockade in the early postnatal mouse brain.

Rapastinel is a novel psychoactive substance that acts as an N-methyl-D-aspartate-receptor (NMDAR) agonist and triggers antidepressant- and antipsychotic-like effects in animal models. However, it is unknown if rapastinel possesses a better side-effect profile than fast-acting glutamatergic antidepressants, like ketamine, which trigger neurotoxicity in the perinatal rodent cortex and protracted schizophrenia-like alterations. Here we found a remarkable neuroprotective effect of rapastinel against apoptosis induced by the NMDAR antagonist MK-801 in comparison to that elicited by clozapine and the mGlu2/3 agonist LY354740. These results suggest the potential therapeutic/prophylactic effect of rapastinel in ameliorating deleterious effects induced by NMDAR blockade during neurodevelopment.

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of γ-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Cortical and Commissural Defects Upon HCF-1 Loss in Nkx2.1-Derived Embryonic Neurons and Glia.

Formation of the cerebral cortex and commissures involves a complex developmental process defined by multiple molecular mechanisms governing proliferation of neuronal and glial precursors, neuronal and glial migration, and patterning events. Failure in any of these processes can lead to malformations. Here, we study the role of HCF‐1 in these processes. HCF‐1 is a conserved metazoan transcriptional co‐regulator long implicated in cell proliferation and more recently in human metabolic disorders and mental retardation. Loss of HCF‐1 in a subset of ventral telencephalic Nkx2.1‐positive progenitors leads to reduced numbers of GABAergic interneurons and glia, owing not to decreased proliferation but rather to increased apoptosis before cell migration. The loss of these cells leads to development of severe commissural and cortical defects in early postnatal mouse brains. These defects include mild and severe structural defects of the corpus callosum and anterior commissure, respectively, and increased folding of the cortex resembling polymicrogyria. Hence, in addition to its well‐established role in cell proliferation, HCF‐1 is important for organ development, here the brain.

Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing

Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown whether these functions are carried out by subsets of microglia during different stages of developmentand adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, early postnatal, and adult mouse brains. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, early postnatal microglia are more heterogeneous. We discovered a proliferative-region-associated microglia (PAM) subset, mainly found in developing white matter, that shares a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.

Signposts for synapses

Neurodevelopment Brain development entails not only positioning the neurons in the right place but also building the correct connections for functional circuits. In the early postnatal mouse brain, Oh et al. discovered how the exact locations for synapse development are specified. The

Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study.

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

Minocycline exacerbates apoptotic neurodegeneration induced by the NMDA receptor antagonist MK-801 in the early postnatal mouse brain.

NMDA receptor (NMDAR) antagonists induce in perinatal rodent cortical apoptosis and protracted schizophrenia-like alterations ameliorated by antipsychotic treatment. The broad-spectrum antibiotic minocycline elicits antipsychotic and neuroprotective effects. Here we tested, if minocycline protects also against apoptosis triggered by the NMDAR antagonist MK-801 at postnatal day 7. Surprisingly, minocycline induced widespread cortical apoptosis and exacerbated MK-801-triggered cell death. In some areas such as the subiculum, the pro-apoptotic effect of minocycline was even more pronounced than that elicited by MK-801. These data reveal among antipsychotics unique pro-apoptotic properties of minocycline, raising concerns regarding consequences for brain development and the use in children.

Paucimannosidic glycoepitopes are functionally involved in proliferation of neural progenitor cells in the subventricular zone.

Protein glycosylation has received much attention due to its multiple functional roles in physiological and pathophysiological conditions. Paucimannose is a common mannosidic N-glycoepitope in invertebrates and plants but has only recently been detected in vertebrates. Herein, we demonstrate the presence of paucimannosidic epitopes specifically in early postnatal neural progenitor cells (NPCs) between postnatal day 0 and 7 in mouse brain suggesting a possible role in the development of NPCs. Paucimannosidic epitopes were also detected in human glioblastoma cells and human macrophages by immunofluorescence and mass spectrometric analysis. Its expression was significantly increased after proliferation arrest indicating its importance in the regulation of cell proliferation. This hypothesis was further strengthened by reduced cell proliferation after the application of paucimannose-reactive Mannitou antibody into culture medium of growing cells. Most interestingly, this reduction in cell proliferation upon the administration of Mannitou antibody could also be observed in vivo in the subventricular zone of early postnatal mouse brain. Taken together, these observations demonstrate that paucimannosylation directly influences cell proliferation in various vertebrate cell types including early postnatal neural stem cells.

Neurons and Glia Modify Receptor Protein-tyrosine Phosphatase ζ (RPTPζ)/Phosphacan with Cell-specific O-Mannosyl Glycans in the Developing Brain

Protein O-mannosylation is a glycan modification that is required for normal nervous system development and function. Mutations in genes involved in protein O-mannosyl glycosylation give rise to a group of neurodevelopmental disorders known as congenital muscular dystrophies (CMDs) with associated CNS abnormalities. Our previous work demonstrated that receptor protein-tyrosine phosphatase ζ (RPTPζ)/phosphacan is hypoglycosylated in a mouse model of one of these CMDs, known as muscle-eye-brain disease, a disorder that is caused by loss of an enzyme (protein O-mannose β-1,2-N-acetylglucosaminyltransferase 1) that modifies O-mannosyl glycans. In addition, monoclonal antibodies Cat-315 and 3F8 were demonstrated to detect O-mannosyl glycan modifications on RPTPζ/phosphacan. Here, we show that O-mannosyl glycan epitopes recognized by these antibodies define biochemically distinct glycoforms of RPTPζ/phosphacan and that these glycoforms differentially decorate the surface of distinct populations of neural cells. To provide a further structural basis for immunochemically based glycoform differences, we characterized the O-linked glycan heterogeneity of RPTPζ/phosphacan in the early postnatal mouse brain by multidimensional mass spectrometry. Structural characterization of the O-linked glycans released from purified RPTPζ/phosphacan demonstrated that this protein is a significant substrate for protein O-mannosylation and led to the identification of several novel O-mannose-linked glycan structures, including sulfo-N-acetyllactosamine containing modifications. Taken together, our results suggest that specific glycan modifications may tailor the function of this protein to the unique needs of specific cells. Furthermore, their absence in CMDs suggests that hypoglycosylation of RPTPζ/phosphacan may have different functional consequences in neurons and glia.

Primary Culture of SVZ-derived Progenitors Grown as Neurospheres

SVZ-derived progenitors grown as neurospheres is a well-known model to study neural stem cell and progenitor functions such as proliferation, differentiation/self-renewal, and migration (Durbec and Rougon, 2001). This protocol is for preparing a culture of SVZ-derived progenitors from 8 early postnatal mouse brains (P0 to P3). One week after cell plating, we can observe round floating neurospheres, each resulting from the clonal expansion of a single EGF/FGF responsive neural progenitor., 作为神经球生长的SVZ衍生的祖细胞是研究神经干细胞和祖细胞功能如增殖，分化/自我更新和迁移的众所周知的模型（Durbec和Rougon，2001）。 该方案用于从8只早期出生后的小鼠脑（P0至P3）制备SVZ衍生祖细胞的培养物。 细胞电镀后一周，我们可以观察到圆形浮游神经球，每个由单个EGF / FGF反应性神经祖细胞的克隆扩增产生。

Development of the principal nucleus trigeminal lemniscal projections in the mouse

The principal sensory (PrV) nucleus-based trigeminal lemniscus conveys whisker-specific neural patterns to the ventroposteromedial (VPM) nucleus of the thalamus and subsequently to the primary somatosensory cortex. Here we examined the perinatal development of this pathway with carbocyanine dye labeling in embryonic and early postnatal mouse brains. We developed a novel preparation in which the embryonic hindbrain and the diencephalon are flattened out, allowing a birds-eye view of the PrV lemniscus in its entirety. For postnatal brains we used another novel approach by sectioning the brain along an empirically determined oblique horizontal angle, again preserving the trigeminothalamic pathway. PrV neurons are born along the hindbrain ventricular zone and migrate radially for a short distance to coalesce into a nucleus adjacent to the ascending trigeminal tract. During migration of the spindle-shaped cell bodies, slender axonal processes grow along the opposite direction towards the floor plate. As early as embryonic day (E) 11, pioneering axons tipped with large growth cones cross the ventral midline and immediately make a right angle turn. By E13 many PrV axons form fascicles crossing the midline and follow a rostral course. PrV axons reach the midbrain by E15 and the thalamus by E17. While the target recognition and invasion occurs prenatally, organization of PrV axon terminals into whisker-specific rows and patches takes place during the first 4 postnatal (P) days. Initially diffuse and exuberant projections in the VPM at P1 coalesce into row and whisker specific terminal zones by P4.

LRRK2 expression is enriched in the striosomal compartment of mouse striatum

In spite of a clear genetic link between Parkinson's disease (PD) and mutations in LRRK2, cellular localization and physiological function of LRRK2 remain debated. Here we demonstrate the immunohistochemical localization of LRRK2 in adult mouse and early postnatal mouse brain development. Antibody specificity is verified by absence of specific staining in LRRK2 knockout mouse brain. Although LRRK2 is expressed in various mouse brain regions (i.e. cortex, thalamus, hippocampus, cerebellum), strongest expression is detected in striatum, whereas LRRK2 protein expression in substantia nigra pars compacta in contrast is low. LRRK2 is highly expressed in striatal medium spiny neurons (MSN) and few cholinergic interneurons. LRRK2 expression is undetectable in other interneurons, oligodendrocytes or astrocytes of the striatum. Interestingly, LRRK2 expression is associated with striosome specific markers (i.e. MOR1, RASGRP1). Analysis of LRRK2 expression during early postnatal development and in LRRK2 knockout mice, demonstrates that LRRK2 is not required for generation or maintenance of the striosome compartment. Comparing LRRK2-WT, LRRK2-R1441G transgenic and non-transgenic mice, changes of LRRK2 expression in striosome/matrix compartments can be detected. The findings rule out a specific requirement of LRRK2 in striosome genesis but suggest a functional role for LRRK2 in striosomes.

Quantitative in vivo 1H spectroscopic imaging of metabolites in the early postnatal mouse brain at 17.6 T

Early postnatal brain maturation is closely connected to local changes of metabolite levels. Spatially resolved in vivo (1)H NMR spectroscopic imaging is applied to follow absolute changes of brain metabolites in early postnatal mouse brain. A short echo time semi LASER (localization by adiabatic selective refocusing) chemical shift imaging (CSI) sequence incorporating weighted k-space averaging was implemented at high magnetic field (17.6 T). In vivo measurements were carried out on postnatal days 5, 8, 12, 16, and 20. In vivo relaxation times T (1) and T (2) were measured using variable repetition times or a CPMG sequence, respectively, combined with LASER single voxel localization. Spectra were obtained with a spatial resolution of (1 x 1) mm(2) in a 1.5 mm slice as early as postnatal day 5. Maturational changes of absolute metabolite concentrations of major metabolites were calculated in four different brain regions. A significant increase of N-acetylaspartate (NAA), total creatine (tCr), and glutamate/glutamine (Glx) concentration was paralleled by a decrease of taurine (Tau) concentration with age (P < 0.05). Differences between brain regions were found for NAA, tCr, and Tau (P < 0.05). Furthermore, in vivo T (1) and T (2) of the four major brain metabolites in adult mice are reported. The implemented semi LASER CSI sequence allows following regional changes of metabolite levels. It is suitable for investigation of local differences in brain metabolism and development.

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.