Morphological Analysis of Neurons and Glia Using Mosaic Analysis with Double Markers.

Mosaic Analysis with Double Markers (MADM) is a powerful genetic method typically used for lineage tracing and to disentangle cell autonomous and tissue-wide roles of candidate genes with single cell resolution. Given the relatively sparse labeling, depending on which of the 19 MADM chromosomes one chooses, the MADM approach represents the perfect opportunity for cell morphology analysis. Various MADM studies include reports of morphological anomalies and phenotypes in the central nervous system (CNS).

Protocol for mapping cell lineage and cell-type identity of clonally-related cells in situ using MADM-CloneSeq.

The lineage relationship of clonally-related cells offers important insights into the ontogeny and cytoarchitecture of the brain in health and disease. Here, we provide a protocol to concurrently assess cell lineage relationship and cell-type identity among clonally-related cells in situ. We first describe the preparation and screening of acute brain slices containing clonally-related cells labeled using mosaic analysis with double markers (MADM).

Protocol for quantitative reconstruction of cell lineage using mosaic analysis with double markers in mice.

The generation of diverse cell types during development is fundamental to brain functions. We outline a protocol to quantitatively assess the clonal output of individual neural progenitors using mosaic analysis with double markers (MADM) in mice. We first describe steps to acquire and reconstruct adult MADM clones in the superior colliculus.

Multipotent progenitors instruct ontogeny of the superior colliculus.

The superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration and is composed of a rich diversity of excitatory and inhibitory neurons and glia. However, the developmental principles directing the generation of SC cell-type diversity are not understood. Here, we pursued systematic cell lineage tracing in silico and in vivo, preserving full spatial information, using genetic mosaic analysis with double markers (MADM)-based clonal analysis with single-cell sequencing (MADM-CloneSeq).

Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry.

Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice and high-resolution phenotyping at the individual cell level. Here, we present a protocol for isolating MADM-labeled cells with high yield for downstream molecular analyses using fluorescence-activated cell sorting (FACS). We describe steps for generating MADM-labeled mice, perfusion, single-cell suspension, and debris removal.

Astroglial Connexin 43 Regulates Synaptic Vesicle Release at Hippocampal Synapses.

Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered.

Physiological synaptic activity and recognition memory require astroglial glutamine.

Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer.

Immunoprecipitation of Ribosome-Bound mRNAs from Astrocytic Perisynaptic Processes of the Mouse Hippocampus.

Translation of distally localized mRNAs is an evolutionary mechanism occurring in polarized cells. It has been observed in astrocytes, whose processes contact blood vessels and synapses. Here, we describe a protocol for the purification of the entire pool of ribosome-bound mRNAs in perisynaptic astrocytic processes (PAPs).

Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning.

Local translation is a conserved mechanism conferring cells the ability to quickly respond to local stimuli. In the brain, it has been recently reported in astrocytes, whose fine processes contact blood vessels and synapses. Yet the specificity and regulation of astrocyte local translation remain unknown.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM).

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically.

Synaptic vesicle generation from activity-dependent bulk endosomes requires a dephosphorylation-dependent dynamin-syndapin interaction.

Activity-dependent bulk endocytosis generates synaptic vesicles (SVs) during intense neuronal activity via a two-step process. First, bulk endosomes are formed direct from the plasma membrane from which SVs are then generated. SV generation from bulk endosomes requires the efflux of previously accumulated calcium and activation of the protein phosphatase calcineurin.

Neuronal Activity Drives Astroglial Connexin 30 in Perisynaptic Processes and Shapes Its Functions.

Astrocytes play key roles in brain functions through dynamic interactions with neurons. One of their typical features is to express high levels of connexins (Cxs), Cx43 and Cx30, the gap junction (GJ)-forming proteins. Cx30 is involved in basic cognitive processes and shapes synaptic and network activities, as shown by recent studies in transgenic animals.

Activity-Dependent Plasticity of Astroglial Potassium and Glutamate Clearance.

Recent evidence has shown that astrocytes play essential roles in synaptic transmission and plasticity. Nevertheless, how neuronal activity alters astroglial functional properties and whether such properties also display specific forms of plasticity still remain elusive. Here, we review research findings supporting this aspect of astrocytes, focusing on their roles in the clearance of extracellular potassium and glutamate, two neuroactive substances promptly released during excitatory synaptic transmission.

Connexons and pannexons: newcomers in neurophysiology.

Connexin hemichannels are single membrane channels which have been traditionally thought to work in pairs to form gap junction channels across two opposing cells. In astrocytes, gap junction channels allow direct intercellular communication and greatly facilitate the transmission of signals. Recently, there has been growing evidence demonstrating that connexin hemichannels, as well as pannexin channels, on their own are open in various conditions.

Synaptic vesicle generation from activity-dependent bulk endosomes requires calcium and calcineurin.

Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during high-frequency stimulation in central nerve terminals. ADBE generates endosomes direct from the plasma membrane, meaning that high concentrations of calcium will be present in their interior due to fluid phase uptake from the extracellular space. Morphological and fluorescent assays were used to track the generation of SVs from bulk endosomes in primary neuronal culture.

Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes.

Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures.

Quantitative analysis of synaptic vesicle pool replenishment in cultured cerebellar granule neurons using FM dyes.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis(1,2). The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP).

Properties of doublecortin-(DCX)-expressing cells in the piriform cortex compared to the neurogenic dentate gyrus of adult mice.

The piriform cortex receives input from the olfactory bulb and (via the entorhinal cortex) sends efferents to the hippocampus, thereby connecting the two canonical neurogenic regions of the adult rodent brain. Doublecortin (DCX) is a cytoskeleton-associated protein that is expressed transiently in the course of adult neurogenesis. Interestingly, the adult piriform cortex, which is usually considered non-neurogenic (even though some reports exist that state otherwise), also contains an abundant population of DCX-positive cells.

Modulation of fate determinants Olig2 and Pax6 in resident glia evokes spiking neuroblasts in a model of mild brain ischemia.

Background And Purpose: Although in vitro studies suggest that non-neurogenic regions of the adult central nervous system potentially contain multipotent parenchymal progenitors, neurons are clearly not replaced in most brain regions after injury. Here, in a well-established model of mild transient brain ischemia, we explored Olig2 antagonism and Pax6 overexpression as potential avenues to redirect endogenous progenitors proliferating in situ toward a neuronal fate.

Methods: Retroviral vectors containing either Pax6 or a strong activator form of the repressor Olig2 (Olig2VP16), ie, a functionally dominant negative form of Olig2, were stereotaxically injected into the lateral striatum at 48 hours after 30 minutes middle cerebral artery occlusion (MCAo)/reperfusion.

Activity-dependent bulk endocytosis and clathrin-dependent endocytosis replenish specific synaptic vesicle pools in central nerve terminals.

Multiple synaptic vesicle (SV) retrieval modes exist in central nerve terminals to maintain a continual supply of SVs for neurotransmission. Two such modes are clathrin-mediated endocytosis (CME), which is dominant during mild neuronal activity, and activity-dependent bulk endocytosis (ADBE), which is dominant during intense neuronal activity. However, little is known about how activation of these SV retrieval modes impact the replenishment of the total SV recycling pool and the pools that reside within it, the readily releasable pool (RRP) and reserve pool.

Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles.

Glycogen synthase kinase 3 (GSK3) is a critical enzyme in neuronal physiology; however, it is not yet known whether it has any specific role in presynaptic function. We found that GSK3 phosphorylates a residue on the large GTPase dynamin I (Ser-774) both in vitro and in primary rat neuronal cultures. This was dependent on prior phosphorylation of Ser-778 by cyclin-dependent kinase 5.

GABAergic activities enhance macrophage inflammatory protein-1alpha release from microglia (brain macrophages) in postnatal mouse brain.

Microglial cells (brain macrophages) invade the brain during embryonic and early postnatal development, migrate preferentially along fibre tracts to their final position and transform from an amoeboid to a ramified morphology. Signals by which the invading microglia communicate with other brain cells are largely unknown. Here, we studied amoeboid microglia in postnatal corpus callosum obtained from 6- to 8-day-old mice.

C1q, the recognition subcomponent of the classical pathway of complement, drives microglial activation.

Microglia, central nervous system (CNS) resident phagocytic cells, persistently police the integrity of CNS tissue and respond to any kind of damage or pathophysiological changes. These cells sense and rapidly respond to danger and inflammatory signals by changing their cell morphology; by release of cytokines, chemokines, or nitric oxide; and by changing their MHC expression profile. We have shown previously that microglial biosynthesis of the complement subcomponent C1q may serve as a reliable marker of microglial activation ranging from undetectable levels of C1q biosynthesis in resting microglia to abundant C1q expression in activated, nonramified microglia.

Mild brain ischemia induces unique physiological properties in striatal astrocytes.

We studied the properties of GFAP-expressing cells in adult mouse striatum using acute brain slices from transgenic animals expressing EGFP under GFAP promoter. Under physiological conditions, two distinct populations of GFAP-EGFP cells could be identified: (1) brightly fluorescent cells had bushy processes, passive membrane properties, glutamate transporter activity, and high gap junction coupling rate typical for classical astrocytes; (2) weakly fluorescent cells were characterized by thin, clearly distinguishable processes, voltage-gated currents, complex responses to kainate, and low coupling rate reminiscent of an astrocyte subtype recently described in the hippocampus. Mild focal cerebral ischemia confers delayed neuronal cell death and astrogliosis in the striatum.

Homocysteine stimulates monocyte chemoattractant protein-1 expression in mesangial cells via NF-kappaB activation.

Hyperhomocysteinemia is regarded as an independent risk factor for cardiovascular disorders. Although renal dysfunction or failure is one of the important factors causing hyperhomocysteinemia, the role of homocysteine (Hcy) in the development of glomerulosclerosis is largely unknown. One of the key events in the pathogenesis of glomerulosclerosis is the infiltration of circulating monocytes into affected glomeruli.