Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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. For example, 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 versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. 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

Dorsal root ganglia neurite outgrowth measured as a function of changes in microelectrode array resistance.

Current research in prosthetic device design aims to mimic natural movements using a feedback system that connects to the patient's own nerves to control the device. The first step in using neurons to control motion is to make and maintain contact between neurons and the feedback sensors. Therefore, the goal of this project was to determine if changes in electrode resistance could be detected when a neuron extended a neurite to contact a sensor. Dorsal root ganglia (DRG) were harvested from chick embryos and cultured on a collagen-coated carbon nanotube microelectrode array for two days. The DRG were seeded along one side of the array so the processes extended across the array, contacting about half of the electrodes. Electrode resistance was measured both prior to culture and after the two day culture period. Phase contrast images of the microelectrode array were taken after two days to visually determine which electrodes were in contact with one or more DRG neurite or tissue. Electrodes in contact with DRG neurites had an average change in resistance of 0.15 MΩ compared with the electrodes without DRG neurites. Using this method, we determined that resistance values can be used as a criterion for identifying electrodes in contact with a DRG neurite. These data are the foundation for future development of an autonomous feedback resistance measurement system to continuously monitor DRG neurite outgrowth at specific spatial locations

Muscarinic Acetylcholine Receptor Localization and Activation Effects on Ganglion Response Properties

This study was undertaken to identify the full complement of muscarinic acetylcholine receptors (mAChRs) in the rabbit retina and to assess mAChR distribution and the functional effects of mAChR activation and blockade on retinal response properties. Understanding the distribution and function of mAChRs in the retina has the potential to provide important insights into the visual changes caused by decreased ACh in the retinas of Alzheimer's patients and the potential visual effects of anticholinergic treatments for ocular diseases

The average change in resistance (MΩ) of each classified electrode after background subtraction for all six experiments (SEM in brackets).

<p>The average change in resistance (MΩ) of each classified electrode after background subtraction for all six experiments (SEM in brackets).</p

High resolution phase contrast photomicrograph of DRG tissue seeded on a microelectrode array after a 2 day culture.

<p>(A). 59 extracellular electrodes were visually classified as having contact with a DRG soma (B) or a DRG neurite (C). For some electrodes it was unclear if there was DRG neurite contact (D) and electrodes devoid of contact with DRG tissue were classified as CLEAN (E). The majority of electrodes classified as CLEAN were located on the opposite side of the array from where the DRG tissue was located (left side of A; scale bar = 200 μm).</p

Electrode resistance values can reflect DRG neurite contact with electrode.

<p>In a single experiment resistance values from electrodes were averaged by classification (DRG neurite, UNCLEAR, or CLEAN). The soma in this experiment was not placed in contact with any electrodes. (A) The change in resistance for electrodes in contact with a DRG neurite (-0.24 ± 0.02 MΩ; n = 16; p < 0.001; F = 69.685) were significantly less than for electrodes classified as UNCLEAR (-0.42 ± 0.02 MΩ; n = 19; p < 0.001) or CLEAN (-0.53 ± 0.01 MΩ; n = 31 electrodes). (B) The distribution of the change in electrode resistances for CLEAN electrodes, UNCLEAR electrodes and electrodes contacted by a DRG neurite were plotted in a frequency curve (0.5 MΩ bins). (C) The average change in resistance of UNCLEAR electrodes was background subtracted across all electrodes in a single experiment. This did not change the relationship between resistance values of electrodes contacted by DRG neurites and CLEAN electrodes. However it allowed for a comparison of resistance values across experiments.</p