| 1. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 2. |
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| 3. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 4. |
- Vlaskovska, Mila, et al.
(författare)
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Opioid effects on 45Ca2+ uptake and glutamate release in rat cerebral cortex in primary culture
- 1997
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 68:2, s. 517-524
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Tidskriftsartikel (refereegranskat)abstract
- Primary cultures of rat cortex, conveniently prepared from newborn animals, were used to study opioid effects on 45Ca2+ uptake and glutamate release. 45Ca2+ uptake, induced by treatment with glutamate or NMDA, was largely blocked by the NMDA antagonist MK-801. K+ depolarization-induced 45Ca2+ uptake was also reduced by MK-801, indicating that the effect was mediated by glutamate release. Direct analysis verified that glutamate, and aspartate, were indeed released. Opioid peptides of the prodynorphin system were also released and these, or other peptides, were functionally active, because naloxone treatment increased glutamate release, as well as the 45Ca2+ uptake induced by depolarization. Opioid agonists, selective for mu-, kappa-, and delta-receptors, inhibited the 45Ca2+ uptake induced by K+ depolarization. The combination of low concentrations of MK-801 and opioid agonists resulted in additive inhibition of K(+)-induced 45Ca2+ uptake. The results indicate that this system may be useful as an in vitro CNS model for studying modulation by opioids of glutamate release and Ca2+ uptake under acute, and perhaps also chronic, opiate treatment.
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| 5. |
- You, Zhi-Bing
(författare)
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Characterization of neuropeptide, monoamine, and amino acid release in the basal ganglia of the rat : neuronal dependence and reciprocal interactions
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Functional interactions in the basal ganglia of rats were characterized with in vivomicrodialysis. The study was mainly focused on the dynorphin and cholecystokinin (CCK) systems. The extracellular levels of both dynorphin B and CCK were found in the pM range inthe neostriatum and substantia nigra under basal conditions. The release of these peptides wasCa++- and K+-dependent. Dynorphin B, as well as GABA, levels in the neotriatum and substantia nigra were significantly decreased following a lesion of the neostriatum, suggesting that these dynorphin B and GABA levels reflect activity of the striato-nigral pathway. CCK levels in the neostriatum were significantly decreased only by combined decortication and callosotomy, confirming histochemical and biochemical evidence for a prominent cortico-striatal CCK innervation of both ipsilateral and contralateral origin. HPLC analysis of microdialysis samples showed that, under basal conditions, dynorphin immunoreactivity represented only dynorphin B peptide, whereas under K-depolarizing conditions, both dynorphin B and its precursor big dynorphin were observed. CCK immunoreactivity measured in the neostriatum mainly represented the sulphated CCK octapeptide (CCK-8). The dopamine (DA) D, receptor agonist, SKF 38393 stimulated the release of dynorphin Band GABA both in the neostriatum and substantia nigra, while the D2 receptor agonist,quinpirole was without effect, suggesting that DA modulates the function of the striato-nigraldynorphin pathway via D, receptors. Ouinpirole inhibited the release of DA both in the neostriatum and substantia nigra, supporting the idea that DA autoreceptors are of the D2 receptor subtype. Local perfusion with CCK-8 (1-100 ~IM) induced a concentration-dependent increase inextracellular dynorphin B and Asp levels, both in neostriatum and substantia nigra, whereas DA levels were only increased in the substantia nigra. A 6-OHDA lesion of the nigro-striatal DA pathway did not affect the effect of CCK-8 on dynorphin B and Asp levels. In the neocortex, local CCK-8 administration increased both Asp and Glu levels. The CCKB antagonist, L-365, 260 (20 mg/kg s. c.), but not the CCKA antagonist, L-364,718 (20 mg/kg s.c.), significantly inhibited the effect of CCK-8 on striatal dynorphin B and Asp levels. In the substantia nigra, however, the effect of CCK-8 on dynorphin B and Asp levels was inhibited by both L-365,260 and L-364,718 to a similar extent, whereas L-364,718, but not L-365,260, prevented the in Çease in nigral DA levels. Thus, it is suggested that CCK-8 modulates dynorphin B and Asp release predominantly via the CCKB receptor subtype in the neostriatum and via both CCKA and CCKB receptor subtypes in the substantia nigra. Systemic as well as local administration of morphine stimulated nigral dynorphin B and GABA release, an effect that was antagonized by a low dose of naloxone (0.2 mg/kg s. c.) which has been shown to block mainly the ll-receptors. Morphine had no effect on DA release.On the other hand, naloxone administered systemically at a high dose (4 mg/kg s. c.) orintracerebrally at high concentrations (10-100 ~lM) increased DA release, which indicates atonic inhibitory role of endogenous opioid peptides on the nigro-striatal DA pathway. The data implicate a previously unknown complexity for opioid regulation of the functions of the basal ganglia. In conclusion, the present study illustrates the complexity in the interactions between neuronal pathways of the basal ganglia. The "classic" nigro-striatal DA pathway is under tonic inhibitory opioid control, perhaps via the dynorphin pathway which can be activated via DAD, receptors. The lesser-known CCK pathways are potentially able to interact at different receptors and sites in a stimulatory manner. The study also provides functional support for a neuronal system releasing Asp but not Glu.
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| 6. |
- You, Zhi-Bing, et al.
(författare)
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Effect of morphine on dynorphin B and GABA release in the basal ganglia of rat
- 1996
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Ingår i: Brain Research. - : Elsevier BV. - 0006-8993 .- 1872-6240. ; 710:1-2, s. 241-248
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Tidskriftsartikel (refereegranskat)abstract
- In vivo microdialysis was used to study the effects of systemic, as well as intracerebral administration of morphine and naloxone on dynorphin B release in neostriatum and substantia nigra of rats. The release of dopamine (DA), gamma-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) was also investigated. Systemic injection of morphine (1 mg/kg s.c.) induced long-lasting increases in extracellular dynorphin B and GABA levels in the substantia nigra, whereas DA, Glu and Asp levels, measured in the same region, were not significantly affected. No effect on striatal neurotransmitter levels was observed following systemic morphine administration. Local perfusion of the substantia nigra with morphine (100 microM) through the microdialysis probe also increased nigral dynorphin B and GABA levels. Perfusion of the neostriatum with morphine (100 microM) significantly increased GABA and dynorphin B levels in the ipsilateral substantia nigra, but no effect was observed locally. Naloxone blocked the effect of systemic morphine administration on nigral dynorphin B and GABA release, already at a dose of 0.2 mg/kg s.c. Naloxone alone, given either systemically (0.2-4 mg/kg s.c.) or intracerebrally (1-100 microM), did not affect dynorphin B or amino acid levels, either in neostriatum or in substantia nigra. However, naloxone produced a concentration-dependent increase in DA levels. The present results indicate that systemic morphine administration stimulates the release of dynorphin B in the substantia nigra, probably by activating the mu-subtype of opioid receptor, since the effect of morphine on nigral dynorphin B and GABA was antagonized by a low dose of naloxone. The increase in extracellular DA levels produced by high concentrations of naloxone, both in neostriatum and substantia nigra, indicates a disinhibitory effect of this drug on DA release, probably via a non-mu subtype of opioid receptors located on nigro-striatal DA neurones.
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