TY - JOUR AB - 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). The analysis of clonally related cell lineages revealed that radial glial progenitors (RGPs) in SC are exceptionally multipotent. Individual resident RGPs have the capacity to produce all excitatory and inhibitory SC neuron types, even at the stage of terminal division. While individual clonal units show no pre-defined cellular composition, the establishment of appropriate relative proportions of distinct neuronal types occurs in a PTEN-dependent manner. Collectively, our findings provide an inaugural framework at the single-RGP/-cell level of the mammalian SC ontogeny. AU - Cheung, Giselle T AU - Pauler, Florian AU - Koppensteiner, Peter AU - Krausgruber, Thomas AU - Streicher, Carmen AU - Schrammel, Martin AU - Özgen, Natalie Y AU - Ivec, Alexis AU - Bock, Christoph AU - Shigemoto, Ryuichi AU - Hippenmeyer, Simon ID - 12875 IS - 2 JF - Neuron SN - 0896-6273 TI - Multipotent progenitors instruct ontogeny of the superior colliculus VL - 112 ER - TY - JOUR AB - 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. We then detail procedures for cell sorting by FACS and downstream analysis. This protocol is suitable for embryonic to adult mice. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).1 AU - Amberg, Nicole AU - Cheung, Giselle T AU - Hippenmeyer, Simon ID - 14683 IS - 1 JF - STAR Protocols KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Neuroscience SN - 2666-1667 TI - Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry VL - 5 ER - TY - JOUR AB - 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. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43−/−), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43−/− mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy. AU - Cheung, Giselle T AU - Chever, Oana AU - Rollenhagen, Astrid AU - Quenech’du, Nicole AU - Ezan, Pascal AU - Lübke, Joachim H. R. AU - Rouach, Nathalie ID - 14783 IS - 8 JF - Cells KW - General Medicine SN - 2073-4409 TI - Astroglial connexin 43 regulates synaptic vesicle release at hippocampal synapses VL - 12 ER - TY - JOUR AB - 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. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes. AU - Cheung, Giselle T AU - Bataveljic, Danijela AU - Visser, Josien AU - Kumar, Naresh AU - Moulard, Julien AU - Dallérac, Glenn AU - Mozheiko, Daria AU - Rollenhagen, Astrid AU - Ezan, Pascal AU - Mongin, Cédric AU - Chever, Oana AU - Bemelmans, Alexis Pierre AU - Lübke, Joachim AU - Leray, Isabelle AU - Rouach, Nathalie ID - 10764 JF - Nature Communications TI - Physiological synaptic activity and recognition memory require astroglial glutamine VL - 13 ER - TY - JOUR AB - 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. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present. AU - Beattie, Robert J AU - Streicher, Carmen AU - Amberg, Nicole AU - Cheung, Giselle T AU - Contreras, Ximena AU - Hansen, Andi H AU - Hippenmeyer, Simon ID - 7815 IS - 159 JF - Journal of Visual Experiments SN - 1940-087X TI - Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM) ER - TY - JOUR AB - 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. However, it is still unknown how calcineurin mediates SV generation. We addressed this question using a series of acute interventions that decoupled the generation of SVs from bulk endosomes in rat primary neuronal culture. This was achieved by either disruption of protein–protein interactions via delivery of competitive peptides, or inhibition of enzyme activity by known inhibitors. SV generation was monitored using either a morphological horseradish peroxidase assay or an optical assay that monitors the replenishment of the reserve SV pool. We found that SV generation was inhibited by, (i) peptides that disrupt calcineurin interactions, (ii) an inhibitor of dynamin I GTPase activity and (iii) peptides that disrupt the phosphorylation-dependent dynamin I–syndapin I interaction. Peptides that disrupted syndapin I interactions with eps15 homology domain-containing proteins had no effect. This revealed that (i) calcineurin must be localized at bulk endosomes to mediate its effect, (ii) dynamin I GTPase activity is essential for SV fission and (iii) the calcineurin-dependent interaction between dynamin I and syndapin I is essential for SV generation. We therefore propose that a calcineurin-dependent dephosphorylation cascade that requires both dynamin I GTPase and syndapin I lipid-deforming activity is essential for SV generation from bulk endosomes. AU - Cheung, Giselle T AU - Cousin, Michael A. ID - 7005 IS - 5 JF - Journal of Neurochemistry SN - 0022-3042 TI - Synaptic vesicle generation from activity‐dependent bulk endosomes requires a dephosphorylation‐dependent dynamin–syndapin interaction VL - 151 ER -