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1.
Fluorescence imaging using FM 1-43 and related styryl dyes has provided invaluable insights into presynaptic function of synapses in culture preparations, but has been limited in use for studying central synapses in vivo or in brain slices, because of excessive fluorescence background due to nonspecific membrane binding of dye. We demonstrate here that focal excitation of FM dyes using two-photon laser-scanning microscopy (TPLSM) provides high resolution of FM 1-43-labeled nerve terminals in brain slices by suppressing out-of-focus background and that a readily releasable pool of vesicles can be selectively and stably labeled by hypertonic shock despite slice diffusion barriers. We find direct TPLSM of FM 1-43-labeled nerve terminals to be superior to treatment of slices with either the fluorescent quencher sulforhodamine 101 or dye scavenger ADVASEP-7 in resolving nerve terminal against background fluorescence, enabling continuous monitoring of vesicular uptake, and release of styryl dyes from individual nerve terminals in brain slices. 相似文献
2.
A simple one-pot-procedure for preparation of protein-reactive, water-soluble merocyanine and cyanine dyes has been developed. The 1-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1) was used as a common starting intermediate. The method allows easy preparation of dyes with chloro- and iodoacetamide side chains for covalent attachment to cysteine. By placing a sulfonato group directly on the dye fluorophore system, dyes with high fluorescence quantum yields in water were generated. Both iodo- and chloroacetamido derivatives were shown to be useful in protein labeling. Less reactive chloroacetamides will be preferential for selective labeling of the most reactive cysteines. 相似文献
3.
The amino acid L-aspartate markedly potentiates the responses elicited by L-glutamate at excitatory neuromuscular synapses in lobster walking limbs. Results are consistent with the idea that aspartate increases the affinity between glutamate and its binding sites in the postsynaptic receptor. Although complications due to release from other amino acid sources are a serious qualification, studies of neurally induced release of glutamate and aspartate suggest that both amino acids are released from excitatory nerve terminals. Experiments comparing the potentiating action of a variety of amino acids with their ability to inhibit glutamate uptake are not supportive of the notion that inhibition of agonist removal is the primary mode of action in the potentiation process. However, this idea, as well as the suggestion that aspartate may induce release of glutamate from extrajunctional entrapment sites, are not ruled out. Indeed, it is likely that the modulatory process embodies a multiplicity of reactions with given ones dominating from preparation to preparation. 相似文献
4.
Catecholamine release and uptake in the mouse prefrontal cortex 总被引:7,自引:0,他引:7
Monitoring the release and uptake of catecholamines from terminals in weakly innervated brain regions is an important step in understanding their importance in normal brain function. To that end, we have labeled brain slices from transgenic mice that synthesize placental alkaline phosphatase (PLAP) on neurons containing tyrosine hydroxylase with antibody-fluorochrome conjugate, PLAP-Cy5. Excitation of the fluorochrome enables catecholamine neurons to be visualized in living tissue. Immunohistochemical fluorescence with antibodies to tyrosine hydroxylase and dopamine beta-hydroxylase revealed that the PLAP labeling was specific to catecholamine neurons. In the prefrontal cortex (PFC), immunohistochemical fluorescence of the PLAP along with staining for dopamine transporter (DAT) and norepinephrine transporter (NET) revealed that all three exhibit remarkable spatial overlap. Fluorescence from the PLAP antibody was used to position carbon-fiber microelectrodes adjacent to catecholamine neurons in the PFC. Following incubation with L-DOPA, catecholamine release and subsequent uptake was measured and the effect of uptake inhibitors examined. Release and uptake in NET and DAT knockout mice were also monitored. Uptake rates in the cingulate and prelimbic cortex are so slow that catecholamines can exist in the extracellular fluid for sufficient time to travel approximately 100 microm. The results support heterologous uptake of catecholamines and volume transmission in the PFC of mice. 相似文献
5.
Neuromodulators that alter the balance between lower-frequency glutamate-mediated excitatory and higher-frequency GABA-mediated inhibitory synaptic transmission are likely to participate in core mechanisms for CNS function and may contribute to the pathophysiology of neurological disorders such as schizophrenia and Alzheimer's disease. Pregnenolone sulfate (PS) modulates both ionotropic glutamate and GABA(A) receptor mediated synaptic transmission. The enzymes necessary for PS synthesis and degradation are found in brain tissue of several species including human and rat, and up to 5 nM PS has been detected in extracts of postmortem human brain. Here, we ask whether PS could modulate transmitter release from nerve terminals located in the striatum. Superfusion of a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes with brief-duration (2 min) pulses of 25 nM PS demonstrates that PS increases the release of newly accumulated [3H]dopamine ([3H]DA), but not [14C]glutamate or [3H]GABA, whereas pregnenolone is without effect. PS does not affect dopamine transporter (DAT) mediated uptake of [3H]DA, demonstrating that it specifically affects the transmitter release mechanism. The PS-induced [3H]DA release occurs via an NMDA receptor (NMDAR) dependent mechanism as it is blocked by D-2-amino-5-phosphonovaleric acid. PS modulates DA release with very high potency, significantly increasing [3H]DA release at PS concentrations as low as 25 pM. This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid. 相似文献
6.
Diolistic labeling is a highly efficient method for introducing dyes into cells using biolistic techniques. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery using a hand-held gene gun, allows non-toxic labeling of multiple cells in both living and fixed tissue. The technique is rapid (labeled cells can be visualized in minutes) and technically undemanding. Here, we provide a detailed protocol for diolistic labeling of cultured human embryonic kidney 293 cells and whole brain using a hand-held gene gun. There are four major steps: (i) coating gold microcarriers with one or more dyes; (ii) transferring the microcarriers into a cartridge to make a bullet; (iii) preparation of cells or intact tissue; and (iv) firing the microcarriers into cells or tissue. The method can be readily adapted to other cell types and tissues. This protocol can be completed in less than 1 h. 相似文献
7.
Sari Izenwasser Henry M. Jacocks John G. Rosenberger Brian M. Cox 《Journal of neurochemistry》1991,56(2):603-610
The effects of both (-)- and (+)-nicotine isomers were examined on in vitro uptake and release of [3H]dopamine in rat striatum. Both isomers inhibited uptake of [3H]dopamine in chopped tissue at concentrations well below those necessary for promoting release of preloaded [3H]dopamine. (-)-Nicotine was more potent than (+)-nicotine both at inhibiting uptake and at promoting release. Unlike other dopamine uptake inhibitors, however, nicotine inhibited only 50% of the total uptake. In the presence of 1 nM nicotine, the residual [3H]dopamine uptake was less sensitive to inhibition by cocaine than uptake in the absence of nicotine. Nicotine did not compete against the binding of [3H]GBR 12935, a selective dopamine uptake inhibitor. The nicotinic receptor agonists carbachol and 1,1-dimethyl-4-phenylpiperazinium iodide also inhibited uptake, whereas the nicotinic antagonists chlorisondamine and mecamylamine blocked nicotine's effect. Thus, the effect of nicotine on dopamine uptake appears to be mediated by a receptor similar to the nicotinic acetylcholine receptor. These receptors do not seem to be on the terminals that are accumulating dopamine, however, since tetrodotoxin prevented the effect of nicotine on [3H]dopamine uptake and nicotine had no effect on uptake in a synaptosomal preparation. 相似文献
8.
The order of proton uptake and release in an aqueous suspension of purple membrane in response to a light flash has been investigated at lowered pH. pH indicator dyes and a flash spectrophotometer were used for the study. At pH 6.6 it was found that the release of protons from the purple membrane precedes uptake, as reported by other investigators. At pH 5.9, 4.9, and 4.1 it was also found that release precedes uptake. These results are not in agreement with those of previous investigators. 相似文献
9.
Adenosine and adenosine analogues inhibited electrically evoked 3H-noradrenaline (3H-NA) release from slices of the rat hippocampus in vitro in a dose -dependent manner in the concentration range 0.01–100 M. L-phenylisopropyladenosine (L-PIA) was more potent than 5′-N-carboxamidoadenosine (NECA), which was more potent than adenosine. The adenosine uptake blocker dipyridamole (3 M) enhanced the effect of exogenous adenosine, and had a slight inhibitory effect per se. The effect of L-PIA on NA release was competitively antagonized by 8-phenyltheopylline; pA2=7.1. Enprophylline (300 M), theophylline (300 M) and 8-phenyltheophylline (1–10 M) enhanced the evoked 3H-NA release per se, while no such enhancement was seen with the non-xanthine phosphodiesterase inhibitor ZK 62.711 (Rolipram) (30 M).It is concluded that adenosine, at physiologically relevant concentrations, inhibits electrically evoked NA release from terminals in the central nervous system. Alkylxanthines increase evoked NA release from hippocampal terminals, wich probably not related to cyclic AMP but may partly involve inhibition of endogenous adenosine acting as a modulator of transmitter release in the hippocampal slice preparation. 相似文献
10.
Targeting liposomes to specific tissues or cells require the unequivocal determination of the uptake of liposomes at the cellular level. The present report describes the preparation of liposomes entrapping a high specific activity of 111In3+-bound inulin, and the potential applications of a multiple labeling technique for characterizing the extent of uptake of liposomes by tissues or different cells in a given tissue in vivo. The labeling method involves the application of the technique of acetylacetone-mediated, ionophoric loading of 111In3+ into liposomes entrapping an inulin derivative to which a strong chelating agent, diethylenetriamine-pentaacetic acid (DTPA), is bound. Subsequent ionophoric removal of the weakly bound 111In3+ by incubating the previously 111In3+-loaded liposomes with 10 mM nitrilotriacetic acid and 100 microM tropolone at room temperature for 20 min results in the preparation of liposomes entrapping 111In3+-DTPA-inulin. Our method of preparation yields net efficiencies of converting 63-78% of the externally added 111In3+ to liposome-entrapped 111In3+-DTPA-inulin. 相似文献
11.
The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity. 相似文献
12.
Processes underlying mechanotransduction and its regulation are poorly understood. Inhibitors of Ca2+-activated K+ channels cause a dramatic increase in afferent output from stretched muscle spindles. We used immunocytochemistry to test for the presence and location of small conductance Ca2+-activated K+ channels (SK1-3) in primary endings of muscle spindles and lanceolate endings of hair follicles in the rat. Tissue sections were double immunolabelled with antibodies to one of the SK channel isoforms and to either synaptophysin (SYN, as a marker of synaptic like vesicles (SLV), present in many mechanosensitive endings) or S100 (a Ca2+-binding protein present in glial cells). SK channel immunoreactivity was also compared to immunolabelling for the Na+ ion channel ASIC2, previously reported in both spindle primary and lanceolate endings. SK1 was not detected in sensory terminals of either muscle spindles or lanceolate endings. SK2 was found in the terminals of both muscle spindles and lanceolate endings, where it colocalised with the SLV marker SYN (spindles and lanceolates) and the satellite glial cell (SGC) marker S100 (lanceolates). SK3 was not detected in muscle spindles; by contrast it was present in hair follicle endings, expressed predominantly in SGCs but perhaps also in the SGC: terminal interface, as judged by colocalisation statistical analysis of SYN and S100 immunoreactivity. The possibility that all three isoforms might be expressed in pre-terminal axons, especially at heminodes, cannot be ruled out. Differential distribution of SK channels is likely to be important in their function of responding to changes in intracellular [Ca2+] thereby modulating mechanosensory transduction by regulating the excitability of the sensory terminals. In particular, the presence of SK2 throughout the sensory terminals of both kinds of mechanoreceptor indicates an important role for an outward Ca2+-activated K+ current in the formation of the receptor potential in both types of ending. 相似文献
13.
The avian iris-ciliary nerve preparation exhibits two distinct choline uptake systems. One component, a sodium dependent, high affinity system Km-2 am and Vmax - 0.5 pmolpin per preparation is confined to nerve terminals. The other component is localized in muscle cells. It is sodium independent and low affinity system (Km - 200 am and Vmax - 16 pmol/min per muscle). The high affinity uptake of choline and the synthesis of ACh in the nerve terminals are coupled. Vmax Ach formation -0.5 pmol/min. is the same as Vmax for choline transport; however. with the external choline concentration equal to that of avian plasma only -50% of choline taken up is converted to ACh. In contrast to the nerve terminals, the cell bodies of the same neurons are deficient in the high affinity uptake-ACh synthesis coupled system. This indicates a nerve terminal membrane specialization related to neuro-transmitter synthesis. 相似文献
14.
《Cytotherapy》2022,24(6):619-628
Background aimsExtracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed.MethodsBy combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia).ResultsThe authors’ analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types.ConclusionsOverall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors’ MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling. 相似文献
15.
Serotonergic transmission is terminated by serotonin transporter (SERT)-mediated uptake following activation of serotonin receptors, several subtypes of which are present in the medial nucleus of the solitary tract (mNTS) at the area postrema level. In this region, serotonin (5HT) is a major modulator of the baroreceptor reflex and also affects gastric motility. This serotonin is derived from multiple sources including local neurons and inputs from raphe and visceral vagal afferents. To determine the relevant functional sites for serotonin uptake in the mNTS, we examined the electron microscopic localization of SERTs using both immunoperoxidase and immunogold labeling in rat brain. In addition, we combined these methods for dual labeling of SERTs and 5HT2A receptors to detect whether the SERT in this region was located near or at a distance from the sites of activation of these G-protein coupled receptors. Intensive SERT immunolabeling was seen on plasma membranes of axons and morphologically heterogeneous axon terminals that formed symmetric or asymmetric synapses on dendrites without detectable 5HT2A immunoreactivity (IR). 5HT2A-IR was, however, located in other nearby neuronal and glial profiles, some of which apposed intensively SERT-labeled terminals or terminals containing lower intensity of SERT immunolabeling. In somatodendritic profiles, co-expression of SERT and 5HT2A receptor immunolabeling was seen near synapses and Golgi lamellae. Our results suggest that in the mNTS 5HT activates 5HT2A receptors at a distance from SERT-mediated uptake sites in diverse cell types including some that express both 5HT2A receptors and SERTs. 相似文献
16.
Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters 总被引:7,自引:4,他引:3
A. Poli A. Contestabile P. Migani L. Rossi C. Rondelli M. Virgili R. Bissoli O. Barnabei 《Journal of neurochemistry》1985,45(6):1677-1686
Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome-enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+-dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40-80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release of excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D-[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+-stimulated release. Kainic acid at 0.1 mM had no clear-cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier-mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high-affinity uptake of L-glutamate and D-aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed. 相似文献
17.
Bhattacharyya BJ Wilson SM Jung H Miller RJ 《American journal of physiology. Cell physiology》2012,302(4):C698-C708
Homozygous ataxic mice (ax(J)) express reduced levels of the deubiquitinating enzyme Usp14. They develop severe tremors by 2-3 wk of age, followed by hindlimb paralysis, and death by 6-8 wk. While changes in the ubiquitin proteasome system often result in the accumulation of ubiquitin protein aggregates and neuronal loss, these pathological markers are not observed in the ax(J) mice. Instead, defects in neurotransmission were observed in both the central and peripheral nervous systems of ax(J) mice. We have now identified several new alterations in peripheral neurotransmission in the ax(J) mice. Using the two-microelectrode voltage clamp technique on diaphragm muscles of ax(J) mice, we observed that under normal neurotransmitter release conditions ax(J) mice lacked paired-pulse facilitation and exhibited a frequency-dependent increase in rundown of the end plate current at high-frequency stimulation (HFS). Combined electrophysiology and styryl dye staining revealed a significant reduction in quantal content during the initial and plateau portions of the HFS train. In addition, uptake of styryl dyes (FM dye) during HFS demonstrated that the size of the readily releasable vesicle pool was significantly reduced. Destaining rates for styryl dyes suggested that ax(J) neuromuscular junctions are unable to mobilize a sufficient number of vesicles during times of intense activity. These results imply that ax(J) nerve terminals are unable to recruit a sufficient number of vesicles to keep pace with physiological rates of transmitter release. Therefore, ubiquitination of synaptic proteins appears to play an important role in the normal operation of the neurotransmitter release machinery and in regulating the size of pools of synaptic vesicles. 相似文献
18.
Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis 总被引:7,自引:1,他引:6
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Using pharmacological (Simpson, L.L., 1980, J. Pharmacol. Exp. Ther. 212:16-21) and autoradiographic techniques (Black, J.D., and J.O. Dolly, 1986, J. Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT-induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with 125I-BoNT. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target. 相似文献
19.
The distribution of the synaptic vesicle-associated phosphoprotein synapsin I after electrical stimulation of the frog neuromuscular junction was investigated by immunogold labeling and compared with the distribution of the integral synaptic vesicle protein synaptophysin. In resting terminals both proteins were localized exclusively on synaptic vesicles. In stimulated terminals they appeared also in the axolemma and its infoldings, which however exhibited a lower synapsin I/synaptophysin ratio with respect to synaptic vesicles at rest. The value of this ratio was intermediate in synaptic vesicles of stimulated terminals, and an increased synapsin I labeling of the cytomatrix was observed. These results indicate that synapsin I undergoes partial dissociation from and reassociation with synaptic vesicles, following physiological stimulation, and are consistent with the proposed modulatory role of the protein in neurotransmitter release. 相似文献
20.
The present study describes a complex array of sensory nerve terminals in the human eyelid. In many respects this pattern of innervation resembles that previously described in the rhesus monkey, but in other respects it is unique to man. The most prominent nerve terminals are a complex array of lanceolate and circular Ruffini and free nerve endings that envelop the eyelashes. In addition, Merkel cells have not been conclusively identified to date in other nonhuman primate nonsinus hairs. The external root sheath collar contains Merkel cells, and dermal Merkel cells have also been identified close to the collar. The anterior cutaneous surface of the eyelid contains small vellus hairs with typical lanceolate, Ruffini, and free nerve endings resembling those of primate facial skin. Scattered Meissner and scant simple corpuscles as well as scattered free nerve endings (FNEs) can be identified on the occlusal surface of the eyelid. Intraepithelial as well as dermal FNEs were most easily identified in this region in areas lacking other corpuscular receptors. Corpuscular receptors are especially common at the occlusal/conjunctival angle. The inner or conjunctival surface of the eyelid appears to be a glandular epithelium, whereas in the rhesus monkey it is stratified squamous epithelium. This epithelium needs additional study. In summary, the present study confirms the unique sensory neural status of the human eyelid and verifies the presence of Ruffini nerve terminals by light and electron microscopy and of free nerve ending terminals at least by light microscopy, as well as a unique pattern of innervation of the human eyelash. 相似文献