Fluorescent Measurement of Synaptic Activity Using FM Dyes in  Dissociated Hippocampal Cultured Neurons

Lazarenko, Roman M.; DelBove, Claire E.; Zhang, Qi

doi:10.21769/bioprotoc.2690

Cited by 7 publications

(5 citation statements)

References 10 publications

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“…Proper dynamics of synaptic vesicles determine effective synaptic transmission. To test whether the altered mPTP flickering caused by paraplegin deficiency influences the exo-endocytic cycle of synaptic vesicles, we exposed Spg7 +/− and Spg7 −/− cortical neurons to the styryl FM dye 1–43 during evoked synaptic activity [ 47 , 48 ].…”

Section: Resultsmentioning

confidence: 99%

“…To gain mechanistic understanding of these processes, we did direct measurements of vesicle release and retrieval by FM1–43 styryl dye (Cat# T3163 Thermo Fisher Scientific, Waltham, MS, USA) since their loading and unloading are reliable measurements for synaptic vesicle release and retrieval in cultured neurons. The imaging procedures that we used have been previously described [47] , [48] . Briefly, presynaptic terminals were labeled by exposure to styryl dye (10 µM FM1–43) during high- K + depolarization (modified Tyrode, 55 mM KCl).…”

Section: Methodsmentioning

confidence: 99%

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Impaired flickering of the permeability transition pore causes SPG7 spastic paraplegia

et al. 2020

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Background Mutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia type 7 (SPG7), a so-far untreatable degenerative disease of the upper motoneuron with still undefined pathomechanism. The intermittent mitochondrial permeability transition pore (mPTP) opening, called flickering, is an essential process that operates to maintain mitochondrial homeostasis by reducing intra-matrix Ca 2+ and reactive oxygen species (ROS) concentration, and is critical for efficient synaptic function. Methods We use a fluorescence-based approach to measure mPTP flickering in living cells and biochemical and molecular biology techniques to dissect the pathogenic mechanism of SPG7. In the SPG7 animal model we evaluate the potential improvement of the motor defect, neuroinflammation and neurodegeneration by means of an mPTP inducer, the benzodiazepine Bz-423. Findings We demonstrate that paraplegin is required for efficient transient opening of the mPTP, that is impaired in both SPG7 patients-derived fibroblasts and primary neurons from Spg7 −/− mice. We show that dysregulation of mPTP opening at the pre-synaptic terminal impairs neurotransmitter release leading to ineffective synaptic transmission. Lack of paraplegin impairs mPTP flickering by a mechanism involving increased expression and activity of sirtuin3, which promotes deacetylation of cyclophilin D, thus hampering mPTP opening. Pharmacological treatment with Bz-423, which bypasses the activity of CypD, normalizes synaptic transmission and rescues the motor impairment of the SPG7 mouse model. Interpretation mPTP targeting opens a new avenue for the potential therapy of this form of spastic paraplegia. Funding Telethon Foundation grant (TGMGCSBX16TT); Dept. of Defense, US Army, grant W81XWH-18–1–0001

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Impaired flickering of the permeability transition pore causes SPG7 spastic paraplegia

et al. 2020

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“…The media was then switched to high potassium (HK; 90 mM KCl, 55 mM NaCl, 10 mM HEPES, 10 mM Glucose, 2.6 mM CaCl2, 1.3 mM MgCl2) or KRH solution for 2 minutes. Individual vesicles were fragmented using ImageJ/FIJI and intensities before and after stimulation were determined as ratios after subtracting background signal and adjusting for image drift (Lazarenko et al, 2018).…”

Section: Synaptic Vesicle Cyclingmentioning

confidence: 99%

Snca-GFP knock-in mice reflect patterns of endogenous expression and pathological seeding

Caputo

Liang

Raabe

et al. 2020

Preprint

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Alpha-synuclein (aSyn) participates in synaptic vesicle trafficking and synaptic transmission, but its misfolding is also strongly implicated in Parkinson's disease (PD) and other neurodegenerative disorders known as synucleinopathies where misfolded aSyn accumulates in different regions of the central and peripheral nervous systems.Although increased aSyn expression levels or altered aggregation propensities likely underlie familial PD with SNCA amplification or mutations, the majority of synucleinopathies arise sporadically, indicating that disease can develop under normal levels of wildtype aSyn. We report here the development and characterization of a mouse line expressing an aSyn-GFP fusion protein under the control of native Snca regulatory elements. Regional and subcellular localization of the aSyn-GFP fusion protein in brains and peripheral tissues of knock-in (KI) mice are indistinguishable from that of wildtype littermates. Importantly, similar to wildtype aSyn, aSyn-GFP disperses from synaptic vesicles upon membrane depolarization, indicating that the tag does not alter normal aSyn dynamics at synapses. In addition, intracerebral injection of aSyn pre-formed fibrils into KI mice induced the formation of aSyn-GFP inclusions with a distribution pattern similar to that observed in wildtype mice, albeit with attenuated kinetics due to the GFP tag. We anticipate that this new mouse model will facilitate in vitro and in vivo studies requiring in situ detection of endogenous aSyn, therefore providing new insights into aSyn function in health and disease. 4 Significance Statement Alpha-synuclein (aSyn) participates in synaptic vesicle function and represents a major component of the Lewy pathology found in Parkinson's and related neurodegenerative diseases. The function of aSyn and the sequence of events leading to its aggregation and neurotoxicity are not fully understood. Here we present a new mouse model in which Enhanced Green Fluorescence Protein (GFP) has been knocked-in at the C-terminal of the Snca gene. The resulting fusion protein shows identical expression and localization to that of wildtype animals, is functional, and is incorporated into pathological aggregates in vitro and in vivo. This new tool allows for monitoring aSyn under a variety of physiological and pathological conditions, and may uncover additional insights into its function and dysfunction.

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“…The imaging setup is the same described for fura-2 calcium assay. FM1-43 fluorescence analyses were performed as described (Lazarenko et al, 2018).…”

Section: Fm1-43 Assaymentioning

confidence: 99%

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis

Pisciottani,

Croci,

Lauria

et al. 2023

Front. Cell. Neurosci.

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Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50–60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.

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Fluorescent Measurement of Synaptic Activity Using FM Dyes in Dissociated Hippocampal Cultured Neurons

Cited by 7 publications

References 10 publications

Impaired flickering of the permeability transition pore causes SPG7 spastic paraplegia

Impaired flickering of the permeability transition pore causes SPG7 spastic paraplegia

Snca-GFP knock-in mice reflect patterns of endogenous expression and pathological seeding

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis

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