Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Control of 5-hydroxytryptamine release in the dorsal raphe nucleus by the noradrenergic system in rat brain. Role of alpha-adrenoceptors

The interactions between the brainstem serotonergic (5-hydroxytryptamine, 5-HT) and noradrenergic (NA) systems are important for the pathophysiology and treatment of affective disorders. We examined the influence of -adrenoceptors on 5-HT and NA release in the rat dorsal raphe nucleus (DR) using microdialysis. 5-HT and NA concentrations in DR dialysates were virtually suppressed by TTX and increased by veratridine. The local and systemic administration of the 1-adrenoceptor antagonist prazosin reduced the DR 5-HT output but not that of NA. The maximal 5-HT reduction induced by local prazosin administration (-78% at 100 M) was more marked than by its systemic administration (-43% at 0.3 mg/kg). The local application of NA and desipramine, to increase the tone on DR 1-adrenoceptors, did not enhance 5-HT release. The local (100 M) or systemic (0.1–1 mg/kg s.c.) administration of clonidine reduced 5-HT and NA release (-48 and -79%, respectively, at 1 mg/kg), an effect reversed by RX-821002, which by itself increased both amines when given systemically. DSP-4 pretreatment prevented the effects of clonidine on 5-HT, suggesting the participation of 2-adrenoceptors on NA elements. Moreover, the systemic effect of clonidine on 5-HT (but not NA) was cancelled by lesion of the lateral habenula and by anesthesia, and was slightly enhanced by cortical transection. These data support the view that 1-adrenoceptors in the DR tonically stimulate 5-HT release, possibly at nearly maximal tone. Likewise, the 5-HT release is modulated by 2-adrenoceptors in NA neurons and in forebrain areas involved in the distal control of 5-HT neurons.Peer reviewe