Imaging Synaptic Activity in Intact Brain and Slices with FM1-43 in C. elegans, Lamprey, and Rat

Kay, Alan R.; Alfonso, Aixa; Alford, Simon; Cline, Hollis T.; Holgado, Andrea; Sakmann, Bert; Snitsarev, Vladislav; Stricker, Thomas; Takahashi, Michiko; Wu, Ling Gang

doi:10.1016/s0896-6273(00)81029-6

Cited by 156 publications

(128 citation statements)

References 23 publications

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“…Mechanisms underlying the interaction of FM2-10 with the guinea pig cough receptor Unlike styryl dye labeling of other cells (Betz et al, 1992;Ryan et al, 1993;Ribchester et al, 1994;Kirchgessner et al, 1996;Hay and Hasser, 1998;Kay et al, 1999;Gale et al, 2001) we found that FM2-10 staining of cough receptors did not require and was not reversed by nerve stimulation. Although labeling was prevented by cooling (suggesting an active process), it was unaffected by removing extracellular calcium, arguing against calciumdependent vesicular transport.…”

Section: Discussionmentioning

confidence: 50%

Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough

Mazzone¹,

Reynolds²,

Mori³

et al. 2009

J. Neurosci.

107

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We have identified a distinct subtype of airway vagal afferent nerve that plays an essential role in regulating the cough reflex. These afferents are exquisitely sensitive to punctate mechanical stimuli, acid, and decreases in extracellular chloride concentrations, but are insensitive to capsaicin, bradykinin, histamine, adenosine, serotonin, or changes in airway intraluminal pressures. In this study we used intravital imaging, retrograde neuronal tracing, and electrophysiological analyses to characterize the structural basis for their peculiar mechanical sensitivity and to further characterize the regulation of their excitability. In completing these experiments, we uncovered evidence for an essential role of an isozyme of Na ϩ -K ϩ ATPase in regulating cough. These vagal sensory neurons arise bilaterally from the nodose ganglia and are selectively and brilliantly stained intravitally with the styryl dye FM2-10. Cough receptor terminations are confined and adherent to the extracellular matrix separating the airway epithelium and smooth muscle layers, a site of extensive remodeling in asthma and chronic obstructive pulmonary disease. The cough receptor terminals uniquely express the ␣ 3 subunit of Na ϩ -K ϩ ATPase. Intravital staining of cough receptors by FM2-10, cough receptor excitability in vitro, and coughing in vivo are potently and selectively inhibited by the sodium pump inhibitor ouabain. These data provide the first detailed morphological description of the peripheral terminals of the sensory nerves regulating cough and identify a selective molecular target for their modulation.

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Section: Discussionmentioning

confidence: 50%

Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough

Mazzone¹,

Reynolds²,

Mori³

et al. 2009

J. Neurosci.

107

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“…Indeed, various markers have been used, such as sulforhodamine (Lichtman et al 1985), fluorescently labeled antibodies (Kraszewski et al 1996;Westphal et al 2008), styryl dyes (i.e., FM dyes; Betz and Bewick 1992), and fluorescent dextrans (Holt et al 2003). Among these markers, FM dyes have proved to be valuable in a number of different preparations, including neuromuscular junctions (NMJ) from the frog (Betz and Bewick 1992), snake (Teng et al 1999), lizard (Lindgren et al 1997), and larval Drosophila (Ramaswami et al 1994); hippocampal neurons in culture (Ryan et al 1993) and acute slice (Zakharenko et al 2001); goldfish retinal bipolar cells (Zenisek et al 2000); Caenorhabditis elegans neurons (Kay et al 1999); rat calyx of Held (de Lange et al 2003); inner and outer hair cells (Griesinger et al 2002;Kaneko et al 2006); and a variety of nonneuronal preparations (for a review, see Cochilla et al 1999). …”

Section: Discussionmentioning

confidence: 99%

“…Another approach to improve the image quality has been to increase the amount of dye that is washed out of the preparation by including the oligosaccharide ADVASEP-7 during the washing steps. ADVASEP-7, which is a derivative of β-cyclodextrin, has a higher affinity for FM dyes than do biological membranes (Kay et al 1999). The addition of ADVASEP-7 to washing solutions greatly reduces background staining, making it possible to image systems such as rat brain and C. elegans slices.…”

Section: Slice Preparationsmentioning

confidence: 99%

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

Hoopmann¹,

Rizzoli²,

Betz³

2011

Cold Spring Harb Protoc

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The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K + ) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. Reagents FM dye(s)Dye concentrations are usually 2-10 µM for FM1-43 or FM4-64, and 25-40 µM for FM2-10. The dyes readily dissolve in water; stocks of 1-5 mM concentration should be prepared and can be stored at 4˚C. Neuronal tissue or cell sample of interest Equipment Imaging setupAn upright epifluorescence microscope equipped with a 40×-60× water-immersion objective is optimal. When working with cell cultures, the advantages of high-resolution oil-immersion objectives (e.g., 100×/1.4 numerical aperture [NA]) can be exploited by using an inverted setup. For FM1-43, maximal fluorescence is at 465-nm excitation/560-nm emission (Henkel et al. 1996); a conventional fluorescein filter set (435-nm excitation) can also be used. Because the major concern in FM imaging is phototoxicity (which appears usually before any clear signs of photobleaching and alters the ability of the preparations to recycle vesicles), keep the illumination to a minimum by including neutral-density filters. For example, when using a 40×/0.75-NA water-immersion objective with a 100-W mercury lamp, 5%-15% transmission is sufficient. On confocal setups, an argon 488-nm laser line is typically used for excitation, but special care has to be taken to avoid phototoxicity. Images are typically acquired by charge-coupled device (CCD) cameras or photomultiplier tubes, which allows for fast acquisition rates and software-based quantitative image analysis.Neuronal stimulator (see Step 3)

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“…Active synapses were labeled with a 2 min application of 10 M FM1-43FX [fixable version of N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] (Invitrogen, Eugene, OR) and 45 mM K ϩ (equimolar substitution for Na ϩ ) in extracellular recording saline containing the following (in mM): 138 NaCl, 4 KCl, 2CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES, and 0.025 D-APV, and 0.001 NBQX, pH 7.25. Cultures were washed for 10 s with extracellular recording saline containing 500 M Advasep-7 (CyDex, Overland Park, KS) to remove nonspecific dye (Kay et al, 1999) and washed in saline alone for 10 min. Cultures were fixed in 4% paraformaldehyde/0.2% glutaraldehyde in PBS for 10 min.…”

Section: Methodsmentioning

confidence: 99%

A Specific Role for Ca²⁺-Dependent Adenylyl Cyclases in Recovery from Adaptive Presynaptic Silencing

Moulder¹,

Jiang²,

Chang³

et al. 2008

J. Neurosci.

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Glutamate generates fast postsynaptic depolarization throughout the CNS. The positive-feedback nature of glutamate signaling likely necessitates flexible adaptive mechanisms that help prevent runaway excitation. We have previously explored presynaptic adaptive silencing, a form of synaptic plasticity produced by ongoing neuronal activity and by strong depolarization. Unsilencing mechanisms that maintain active synapses and restore normal function after adaptation are also important, but mechanisms underlying such presynaptic reactivation remain unexplored. Here we investigate the involvement of the cAMP pathway in the basal balance between silenced and active synapses, as well as the recovery of baseline function after depolarization-induced presynaptic silencing. Activation of the cAMP pathway activates synapses that are silent at rest, and pharmacological inhibition of cAMP signaling silences basally active synapses. Adenylyl cyclase (AC) 1 and AC8, the major Ca 2ϩ -sensitive AC isoforms, are not crucial for the baseline balance between silent and active synapses. In cells from mice doubly deficient in AC1 and AC8, the baseline percentage of active synapses was only modestly reduced compared with wild-type synapses, and forskolin unsilencing was similar in the two genotypes. Nevertheless, after strong presynaptic silencing, recovery of normal function was strongly inhibited in AC1/AC8-deficient synapses. The entire recovery phenotype of the double null was reproduced in AC8-deficient but not AC1-deficient cells. We conclude that, under normal conditions, redundant cyclase activity maintains the balance between presynaptically silent and active synapses, but AC8 plays a particularly important role in rapidly resetting the balance of active to silent synapses after adaptation to strong activity.

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Imaging Synaptic Activity in Intact Brain and Slices with FM1-43 in C. elegans, Lamprey, and Rat

Cited by 156 publications

References 23 publications

Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough

Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

A Specific Role for Ca²⁺-Dependent Adenylyl Cyclases in Recovery from Adaptive Presynaptic Silencing

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Imaging Synaptic Activity in Intact Brain and Slices with FM1-43 in C. elegans, Lamprey, and Rat

Cited by 156 publications

References 23 publications

Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough

Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

A Specific Role for Ca2+-Dependent Adenylyl Cyclases in Recovery from Adaptive Presynaptic Silencing

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A Specific Role for Ca²⁺-Dependent Adenylyl Cyclases in Recovery from Adaptive Presynaptic Silencing