RESUMO
Associational/commissural CA3-CA3 synapses define the recurrent CA3 network that generates the input to CA1 pyramidal neurons. We quantified the fine structure of excitatory synapses in the stratum radiatum of the CA3d area in adult wild type (WT) and fibroblast growth factor 22 knock-out (FGF22KO) mice by using serial 3D electron microscopy. WT excitatory CA3 synapses are rather small yet range 10 fold in size. Spine size, however, was small and uniform and did not correlate with the size of the synaptic junction. To reveal mechanisms that regulate presynaptic structure, we investigated the role of FGF22, a target-derived signal specific for the distal part of area CA3 (CA3d). In adult FGF22KO mice, postsynaptic properties of associational CA3 synapses were unaltered. Presynaptically, the number of synaptic vesicles (SVs), the bouton volume, and the number of vesicles in axonal regions (the super pool) were reduced. This concurrent decrease suggests concerted control by FGF22 of presynaptic size. This hypothesis is supported by the finding that WT presynapses in the proximal part of area CA3 (CA3p) that do not receive FGF22 signaling in WT mice were smaller than presynapses in CA3d in WT but of comparable size in CA3d of FGF22KO mice. Docked SV density was decreased in CA1, CA3d, and CA3p in FGF22KO mice. Because CA1 and CA3p are not directly affected by the loss of FGF22, the smaller docked SV density may be an adaptation to activity changes in the CA3 network. Thus, docked SV density potentially is a long-term regulator for the synaptic release probability and/or the strength of short-term depression in vivo.
Assuntos
Região CA3 Hipocampal/metabolismo , Fatores de Crescimento de Fibroblastos/deficiência , Terminações Pré-Sinápticas/metabolismo , Sinapses/metabolismo , Animais , Região CA3 Hipocampal/ultraestrutura , Feminino , Fatores de Crescimento de Fibroblastos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terminações Pré-Sinápticas/ultraestrutura , Sinapses/ultraestruturaRESUMO
The synaptic vesicle (SV) cycle was initially discovered at the neuromuscular junction using electron microscopy (EM) analysis.(1) With the introduction of fluorescent probes that are able to monitor real-time cellular events in live cells, EM analysis was pushed to the side lines because it could not provide meaningful kinetic analyses of the various steps in the synaptic vesicle cycle.
RESUMO
Cells communicate via endo- and exocytosis with their environment and neighboring cells. At synapses of the nervous system, fast exocytosis is coupled to fast endocytosis, which forms the basis for neurotransmitter release. The introduction of the unique fluorescent FM dyes allowed the monitoring of fast synaptic vesicle exo-endocytic cycling during live imaging sessions and after photoconversion of FM dyes into an electron-dense diaminobenzidine polymer synaptic vesicle cycling can be studied in the electron microscope. This protocol describes FM dye labeling of synaptic vesicles of cultured hippocampal neurons and photoconversion of the fluorescent synaptic vesicles for analysis in the electron microscope (EM).
Assuntos
Endocitose , Corantes Fluorescentes/química , Microscopia Eletrônica , Compostos de Piridínio/química , Compostos de Amônio Quaternário/química , Animais , Astrócitos/química , Astrócitos/citologia , Células Cultivadas , Hipocampo/citologia , Neurônios/química , Neurônios/citologia , Processos Fotoquímicos , Ratos , Coloração e Rotulagem , Fatores de TempoRESUMO
A modern electron microscopic approach to the investigation of the structural organization of proteins and subcellular structures demands the use of molecular genetic techniques. The successful implementation of genetic techniques is closely tied to a reporter gene such as the green fluorescent protein (GFP). Although GFP has been widely used for light microscopy, it has many limitations for use in electron microscopy. In the search for a reporter gene for electron microscopy, interest in the use of horseradish peroxidase (HRP) DNA has recently increased, and several studies already have proven the feasibility of HRP expression in mammalian cells. Here, we describe a protocol that uses a HRP chimera to label the endoplasmic reticulum of HEK cells.