ABSTRACT
We recently established a simple and versatile adeno-associated virus (AAV) induction approach that enables dense (>90% labeled neurons) and cortical-wide Ca2+ sensor expression. Here, we describe the stepwise protocol for neonatal AAV injection of a Ca2+ sensor. We also detail the steps for subsequent craniotomy to generate a chronic cranial window, followed by wide-field two-photon Ca2+ imaging in an awake mouse. This protocol serves as an alternative to the use of transgenic animals and offers translatable options for cortical-wide experiments. For complete details on the use and execution of this protocol, please refer to Ota et al. (2021).
Subject(s)
Cerebral Cortex/diagnostic imaging , Dependovirus/genetics , Optical Imaging/methods , Animals , Calcium/metabolism , Craniotomy , Female , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Injections , Male , Mice , Skull/surgeryABSTRACT
In the version of this article initially published, a Supplementary Fig. 6f was cited in the last paragraph of the Results. No such panel exists; the citation has been deleted. The error has been corrected in the HTML and PDF versions of the article.
ABSTRACT
A localized transcriptome at the synapse facilitates synapse-, stimulus- and transcript-specific local protein synthesis in response to neuronal activity. While enzyme-mediated mRNA modifications are known to regulate cellular mRNA turnover, the role of these modifications in regulating synaptic RNA has not been studied. We established low-input m6A-sequencing of synaptosomal RNA to determine the chemically modified local transcriptome in healthy adult mouse forebrains and identified 4,469 selectively enriched m6A sites in 2,921 genes as the synaptic m6A epitranscriptome (SME). The SME is functionally enriched in synthesis and modulation of tripartite synapses and in pathways implicated in neurodevelopmental and neuropsychiatric diseases. Interrupting m6A-mediated regulation via knockdown of readers in hippocampal neurons altered expression of SME member Apc, resulting in synaptic dysfunction including immature spine morphology and dampened excitatory synaptic transmission concomitant with decreased clusters of postsynaptic density-95 (PSD-95) and decreased surface expression of AMPA receptor subunit GluA1. Our findings indicate that chemical modifications of synaptic mRNAs critically contribute to synaptic function.
Subject(s)
Adenosine/analogs & derivatives , Prosencephalon/metabolism , Synapses/physiology , Synaptic Transmission/physiology , Adenosine/genetics , Adenosine/metabolism , Animals , Mice , TranscriptomeABSTRACT
Iron is an essential transition metal species for all living organisms and plays various physiologically important roles on the basis of its redox activity; accordingly, the disruption of iron homeostasis triggers oxidative stress and cellular damage. Therefore, cells have developed sophisticated iron-uptake machinery to acquire iron while protecting cells from uncontrolled oxidative damage during the uptake process. To examine the detailed mechanism of iron uptake while controlling the redox status, it is necessary to develop useful methods with redox state selectivity, sensitivity, and organelle specificity to monitor labile iron, which is weakly bound to subcellular ligands. Here, we report the development of Mem-RhoNox to monitor local Fe(II) at the surface of the plasma membrane of living cells. The redox state-selective fluorescence response of the probe relies on our recently developed N-oxide strategy, which is applicable to fluorophores with dialkylarylamine in their π-conjugation systems. Mem-RhoNox consists of the N-oxygenated rhodamine scaffold, which has two arms, both of which are tethered with palmitoyl groups as membrane-anchoring domains. In an aqueous buffer, Ac-RhoNox, a model compound of Mem-RhoNox, shows a fluorescence turn-on response to the Fe(II) redox state-selectively. An imaging study with Mem-RhoNox and its derivatives reveals that labile Fe(II) is transiently generated during the major iron-uptake pathways: endocytotic uptake and direct transport. Furthermore, Mem-RhoNox is capable of monitoring endosomal Fe(II) in primary cultured neurons during endocytotic uptake. This report is the first example that identifies the generation of Fe(II) over the course of cellular iron-uptake processes.
Subject(s)
Fluorescent Dyes/chemistry , Iron/analysis , Rhodamines/chemistry , Animals , Cell Membrane/metabolism , Endocytosis , Fluorescent Dyes/chemical synthesis , Hep G2 Cells , Hippocampus/metabolism , Humans , Iron/chemistry , Iron/metabolism , Mice, Inbred ICR , Oxidation-Reduction , Rhodamines/chemical synthesisABSTRACT
Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.
