56 research outputs found
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. 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 proteinâprotein 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 Iâsyndapin 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
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 utilised to track the generation of SVs from bulk endosomes in primary neuronal culture. This process was functionally uncoupled from both SV exocytosis and plasma membrane retrieval events by intervening only after SV fusion and endocytosis were completed. Either intracellular (BAPTA-AM) or intra-endosomal (Rhod-dextran) calcium chelation inhibited SV generation from bulk endosomes, indicating calcium efflux from this compartment is critical for this process. The V-type ATPase antagonist bafilomycin A1 also arrested SV generation from bulk endosomes indicating endosomal acidification may be required for calcium efflux. Finally pharmacological inhibition of the calcium-dependent protein phosphatase calcineurin blocked endosomal SV generation, identifying it as a key downstream effector in this process. These results reveal a novel and key role for the fluid phase uptake of extracellular calcium and its subsequent efflux in the SV lifecycle
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 exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli
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. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes, and that both brefeldin A and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor proteins 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor protein 1 and 3 indicated that they generated the same population of SVs. Thus adaptor protein complex 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis
Metastatic cervical paravertebral solitary fibrous tumor detected by fluorodeoxyglucose positron emission tomography-computed tomography.
Solitary fibrous tumor/hemangiopericytomas (SFT/HPC) are soft tissue tumors that can arising from the abomen, pleura, head and neck, or extremities. We report an unusual case of recurrent hemangiopericytoma in a 67-year-old female presenting with a painless and palpable mass within her right posterior neck. Eight years after initial resection of the mass, a follow-up MRI showed multiple enlarging calvarial lesions. A whole body FDG-PET/CT revealed not only hypermetabolic calvarial lesions but also numerous hypermetabolic axillary node and osseous metastases. Though the majority of these soft tissue tumors exhibit benign behavior and carry a favorable prognosis, patients with these slow growing tumors are at risk for local recurrence and distant metastases which demonstrate substantial FDG avidity. Additional studies are needed to clarify the role of whole body FDG-PET/CT in the surveillance of SFT/HPC to detect recurrent or metastatic lesions
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. We asked how similar these cells would be to DCX-positive cells in the course of adult hippocampal neurogenesis. Using BAC-generated transgenic mice that express GFP under the DCX promoter, we studied DCX-expression and electrophysiological properties of DCX-positive cells in the mouse piriform cortex in comparison with the dentate gyrus. While one class of cells in the piriform cortex indeed showed features similar to newly generated immature granule neurons, the majority of DCX cells in the piriform cortex was mature and revealed large Na+ currents and multiple action potentials. Furthermore, when proliferative activity was assessed, we found that all DCX-expressing cells in the piriform cortex were strictly postmitotic, suggesting that no DCX-positive âneuroblastsâ exist here as they do in the dentate gyrus. We conclude that DCX in the piriform cortex marks a unique population of postmitotic neurons with a subpopulation that retains immature characteristics associated with synaptic plasticity. DCX is thus, per se, no marker of neurogenesis but might be associated more broadly with plasticity
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. 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
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. Using both acute inhibition with pharmacological antagonists and silencing of expression with short hairpin RNA, we found that GSK3 was specifically required for activity-dependent bulk endocytosis (ADBE) but not clathrin-mediated endocytosis. Moreover we found that the specific phosphorylation of Ser-774 on dynamin I by GSK3 was both necessary and sufficient for ADBE. These results demonstrate a presynaptic role for GSK3 and they indicate that a protein kinase signaling cascade prepares synaptic vesicles for retrieval during elevated neuronal activity
Enriched Monolayer Precursor Cell Cultures from Micro-Dissected Adult Mouse Dentate Gyrus Yield Functional Granule Cell-Like Neurons
BACKGROUND: Stem cell cultures are key tools of basic and applied research in Regenerative Medicine. In the adult mammalian brain, lifelong neurogenesis originating from local precursor cells occurs in the neurogenic regions of the hippocampal dentate gyrus. Despite widespread interest in adult hippocampal neurogenesis and the use of mouse models to study it, no protocol existed for adult murine long-term precursor cell cultures with hippocampus-specific differentiation potential. METHODOLOGY/PRINCIPAL FINDINGS: We describe a new strategy to obtain serum-free monolayer cultures of neural precursor cells from microdissected dentate gyrus of adult mice. Neurons generated from these adherent hippocampal precursor cell cultures expressed the characteristic markers like transcription factor Prox1 and showed the TTX-sensitive sodium currents of mature granule cells in vivo. Similar to granule cells in vivo, treatment with kainic acid or brain derived neurotrophic factor (BDNF) elicited the expression of GABAergic markers, further supporting the correspondence between the in vitro and in vivo phenotype. When plated as single cells (in individual wells) or at lowest density for two to three consecutive generations, a subset of the cells showed self-renewal and gave rise to cells with properties of neurons, astrocytes and oligodendrocytes. The precursor cell fate was sensitive to culture conditions with their phenotype highly influenced by factors within the media (sonic hedgehog, BMP, LIF) and externally applied growth factors (EGF, FGF2, BDNF, and NT3). CONCLUSIONS/SIGNIFICANCE: We report the conditions required to generate adult murine dentate gyrus precursor cell cultures and to analyze functional properties of precursor cells and their differentiated granule cell-like progeny in vitro
Gliazellen im sich entwickelnden und verletzten Gehirn - Eigenschaften und Funktionen
Emerging findings demonstrate that in the central nervous system, glial cells
interact with neuronal networks and thereby modulate brain activities. In
particular, microglia and astrocytes respond rapidly towards the environment
with morphological and physiological changes. In two separate projects, the
functions of invading microglia in the developing brain (Project 1) and
properties of astrocytes in response to mild focal ischemia (Project 2) were
investigated: Project 1: Microglia invade the brain during early stages of
development and migrate along fiber tracts to their final destinations where
they become resident. However, specific signals through which they communicate
with the developing brain are yet largely unknown. The present study utilized
ameboid microglia at the corpus callosum of postnatal mouse acute brain slices
as a model for invading microglia. Their response towards GABAA receptor
activities, which is essential for trophic actions of GABA in brain
development, was studied. Whole cell patch-clamp experiments revealed that
muscimol, a specific agonist for GABAA receptor, triggered a transient
increase in K+ conductance in ameboid microglia. Such response was
progressively lost in single cells lifted up from slice surface and was
observed in cultured microglia only in close vicinity to brain slices
suggesting that it was indirect. Muscimol also stimulated GABAA receptors on
macroglia and neurons in postnatal brain leading to an elevation of
extracellular K+ concentration ([K+]0). In addition, an experimental increase
of [K+]0 mimicked muscimol-induced current response in microglia. These
results indicated that invading microglia in early postnatal development sense
GABAergic activities indirectly via sensing changes in [K+]0. This in turn
stimulated the release of a chemokine, macrophage inflammatory protein-1
alpha, from microglia in vitro and from postnatal brain slices; while the
release of a number of other cytokines, microglial chemotaxis and
proliferation were not affected. Moreover, in adult brain slices, muscimol
only led to a small increase in [K+]0 which failed to elicit current response
in ramified microglia suggesting functional significance specific for the
developing brain. Concurrently, motility and intracellular Ca2+ level of
ameboid microglia were also enhanced upon GABAA receptor stimulation. Project
2: There is at present no consensus about the beneficial or detrimental roles
of astrocytes in brain injuries. Using adult transgenic mice expressing
enhanced green fluorescent protein under the glial fibrillary acidic protein
(GFAP) promoter, astrocytes in the striatum were characterized before and
after mild focal ischemia. In healthy striatum, two types of GFAP positive
astrocytes with distinctive properties could be identified; 1) brightly
fluorescent cells were characterized by bushy processes, passive membrane
properties, glutamate transporter activity, and formed large gap junction
coupling network; 2) weakly fluorescent cells generally had thin and clearly
distinguishable processes, voltage-gated currents, complex current responses
to kainate, and low coupling rate. While the brightly fluorescent cells have
properties typical for classical astrocytes, the weakly fluorescent ones
resemble a glial cell subtype recently described in the hippocampus. To
investigate properties of these cell populations upon brain injury, short
middle cerebral artery occlusion (MCAo) followed by reperfusion which leads to
delayed neuronal cell death and astrogliosis in the striatum was used as a
model for mild focal ischemia. In response to MCAo/reperfusion, brightly
fluorescent cells became dominant over days within the ischemic lesion and the
majority of these cells expressed voltage-gated channels and showed complex
responses to kainate. Interestingly, they had high coupling rate which even
exceeded that of control brightly fluorescent cells. Conversely, a minority of
cells found after ischemia had passive membrane properties and decreased
coupling rate. Taken together, findings from these projects demonstrated
properties of glial cells in the developing and injured brains suggesting
possible functional significance. Invading microglia, although they did not
express functional GABAA receptor subtype, could sense GABAergic activities in
neighboring cells of the developing brain via [K+]0 increase. This modulated
microglial properties which may contribute to white matter development.
Striatal astrocytes, on the other hand, underwent distinct pathophysiological
changes after ischemic insults and constituted a novel physiological phenotype
which formed large syncytium. The delayed and long-lasting effects suggested
possible neuroprotective functions of these reactive astrocytes.JĂŒngere Forschungsergebnisse zeigen, dass Gliazellen eine wichtige Rolle im
Zentralnervensystem spielen, die darin besteht, dass sie mit neuronalen
Netzwerken interagieren und somit HirntÀtigkeiten modulieren. Besonders
Mikroglia und Astrozyten reagieren unmittelbar mit aktiver Ănderung ihrer
Zellmorphologie und â physiologie auf Reize ihrer Umgebung. Somit wurde in
zwei getrennten Projekten die Funktionen einwandernder Mikroglia im sich
entwickelnden Gehirn (Projekt 1) sowie die Eigenschaften von Astrozyten als
Reaktion auf Fokale IschÀmie (Projekt 2) untersucht: Projekt 1: Mikroglia
dringen wĂ€hrend frĂŒher Entwicklungsstadien in das Gehirn ein und wandern
entlang von Fasertrakten zu ihrem Zielort, an dem sie in die ruhende Form
ĂŒbergehen. Dennoch sind die Signale, durch die sie mit dem sich entwickelnden
Gehirn kommunizieren, noch immer weitgehend unbekannt. Die hier vorgelegte
Studie nutzte amöboide Mikroglia aus dem Corpus callosum akuter Hirnschnitte
aus postnatalen MĂ€usen als ein Modell fĂŒr einwandernde Mikroglia. Es wurde
deren Reaktion auf GABAA RezeptoraktivitĂ€t, welche essentiell fĂŒr die
trophische Wirkung von GABA wÀhrend der Hirnentwicklung ist, untersucht. Whole
cell patch clamp Experimente erbrachten den Beweis, dass Muscimol, ein
spezifischer GABAA Rezeptor-Agonist, eine transiente Erhöhung der
KaliumleitfÀhigkeit in amöboiden Mikrogliazellen erzeugt. Diese Reaktion
verschwandt sukzessive als einzelne Mikroglia ĂŒber die SchnittoberflĂ€che
angehoben wurden. Ebenso war die Reaktion in Kultur nur in unmittelbarer NĂ€he
zu einem Hirnschnitt zu beobachten, was auf einen indirekten Effekt schlieĂen
lÀsst. Muscimol aktivierte im postnatalen Gehirn GABAA Rezeptoren auf
benachbarten Makroglia sowie auf Neuronen, was zu einer Erhöhung der
extrazellulĂ€ren Kaliumkonzentration ([K+]O) fĂŒhrte. ZusĂ€tzlich konnte eine
experimentelle [K+]O Erhöhung den Effekt von Muscimol imitieren. Diese
Ergebnisse zeigten, dass eindringende Mikroglia wĂ€hrend der frĂŒhen postnatalen
Entwicklung GABAerge AktivitĂ€t indirekt durch Ănderungen der [K+]O
detektieren. Dies wiederum stimulierte die Freisetzung eines Chemokins namens
macrophage inflammatory protein-1 alpha sowohl aus postnatalen Hirnschnitten
als auch aus Mikrogliazellen in Kultur; die Freisetzung anderer Zytokine, die
mikrogliale Chemotaxis sowie Proliferation wurden nicht beeinflusst. Des
weiteren fĂŒhrte Muscimol in Hirnschnitten von adulten MĂ€usen nur zu einem
geringen Anstieg der [K+]O, welcher nicht in der Lage war, eine Stromantwort
in ramifizierten Mikroglia hervorzurufen. Dies legt eine funktionelle
Signifikanz spezifisch fĂŒr das sich entwickelnde Gehirn nahe. Gleichzeitig
fĂŒhrte die GABAA Rezeptor Stimulation zu einer Erhöhung der MotilitĂ€t und der
intrazellulÀren Kalziumkonzentrationen amöboider Mikroglia. Projekt 2: Derzeit
besteht keine Einigkeit bezĂŒglich der förderlichen oder schĂ€dlichen Rolle von
Astrozyten bei Hirnverletzungen. Unter Zuhilfenahme adulter transgener MĂ€use,
die das enhanced green fluorescence protein (EGFP) unter dem Promotor von
glial fibrillary acidic protein (GFAP) exprimieren, wurden Populationen von
Astrozyten im Striatum vor und nach Fokaler IschÀmie untersucht. In akuten
Schnitten, die aus gesunden Hirnen gewonnen wurden, konnten zwei Typen von
GFAP-positiven Astrozyten im Striatum ausgemacht werden. 1) Hell
fluoreszierende Zellen, charakterisiert durch buschige FortsÀtze, passive
Membraneigenschaften, Glutamattransporter-AktivitÀt und Kopplung durch Gap
Junction Netzwerke. 2) Schwach fluoreszierende Zellen mit dĂŒnnen und klar zu
identifizierenden FortsÀtzen, spannungsaktivierten Strömen, einer komplexen
Stromantwort auf Kainat und einer geringen Kopplungsrate. WĂ€hrend die hell
fluoreszierenden Zellen Charakteristika klassischer Astrozyten zeigten, hatten
die schwach fluoreszierenden Zellen Ăhnlichkeit mit einem kĂŒrzlich im
Hippocampus beschriebenen astrozytÀren Subtyp. Um die Eigenschaften dieser
Zellpopulationen in Reaktion auf Hirnverletzungen zu untersuchen, wurden kurze
Okklusionen der mittleren zerebralen Arterie (MCAo), gefolgt von Reperfusion,
durchgefĂŒhrt. Dies fĂŒhrte zu verzögerten neuronalen Zelltod und Astrogliose im
Striatum und diente als Modell fĂŒr Fokale IschĂ€mie. Als Antwort auf
MCAo/Reperfusion zeigte sich, dass stark fluoreszierende Zellen im Laufe
mehrerer Tage dominant im Bereich der ischÀmischen LÀsion wurden. Die Mehrheit
dieser Zellen exprimierte spannungsaktivierte KanÀle und zeigte komplexe
Antworten auf Kainat. Interessanterweise zeigten diese Zellen eine höhere
Kopplungsrate als Kontrollzellen mit starker Fluoreszenz. Eine Minderheit der
Zellen hingegen zeigte passive Membraneigenschaften und wies im Vergleich zu
passiven Kontrollzellen eine verminderte Kopplungsrate auf. Zusammengefasst
zeigen die Ergebnisse aus dieser Studie Eigenschaften von Gliazellen im sich
entwickelnden und verletzten adulten Gehirn, die eine funktionelle Signifikanz
nahelegen. Einwandernde Mikroglia können GABAerge AktivitÀten in benachbarten
Zellen des sich entwickelnden Gehirns teilweise detektieren obwohl sie keine
funktionellen GABAA Rezeptoren exprimieren. Dies findet durch eine Erhöhung
der [K+]O statt, welche die mikroglialen Eigenschaften moduliert, was
möglicherweise zur Entwicklung der weiĂen Substanz beitrĂ€gt. Andererseits
unterlagen striatale Astrozyten verschiedenen pathophysiologischen Ănderungen
nach einer IschÀmie und stellten einen neuen physiologischen PhÀnotyp dar, der
ein groĂes Synzytium bildete. Der verzögerte und lang anhaltende Effekt legt
eine mögliche neuroprotektive Funktion reaktiver Astrozyten nahe
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