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

Organisation of sensitisation of hind limb withdrawal reflexes from acute noxious stimuli in the rabbit

Spatial aspects of central sensitisation were investigated by studying the effects on three hind limb withdrawal reflexes of an acute noxious stimulus (20 % mustard oil) applied to a number of locations around the body in decerebrate and in anaesthetised rabbits. Reflex responses to electrical stimulation of the toes were recorded from the ankle flexor tibialis anterior (TA) and the knee flexor semitendinosus (ST), whereas responses to stimulation of the heel were recorded from the ankle extensor medial gastrocnemius (MG). In non-spinalised, decerebrated, pentobarbitone-sedated preparations, flexor reflexes were facilitated significantly from sites on the plantar surface of the ipsilateral foot but were either inhibited or unaffected by stimulation of sites away from this location. The heel–MG reflex was facilitated from the ipsilateral heel and was inhibited from a number of ipsilateral, contralateral and off-limb sites. In decerebrated, spinalised, pentobarbitone-sedated animals, mustard oil applied to any site on the ipsilateral hind limb enhanced both flexor reflexes, whereas the MG reflex was enhanced only after stimulation at the ipsilateral heel and was inhibited after stimulation of the toe tips or TA muscle. Mustard oil on the contralateral limb had no effect on any reflex. In rabbits anaesthetised with pentobarbitone and prepared with minimal surgical interference, the sensitisation fields for the heel–MG and toes–TA reflexes were very similar to those in non-spinal decerebrates whereas that for toes–ST was more like the pattern observed in spinalised animals. In no preparation was sensitisation or inhibition of reflexes related to the degree of motoneurone activity generated in direct response to the sensitising stimulus. This study provides for the first time a complete description of the sensitisation fields for reflexes to individual muscles. Descending controls had a marked effect on the area from which sensitisation of flexor reflexes could be obtained, as the sensitisation fields for the flexor reflexes evoked from the toes were larger in spinalised compared to decerebrated, non-spinalised animals. The intermediate sizes of sensitisation fields in anaesthetised animals suggests that the area of these fields can be dynamically controlled from the brain. On the other hand, the sensitisation field for the heel–MG reflex varied little between preparations and appears to be a function of spinal neurones

Imparied capsaicin and neurokinin-evoked colonic motility in inflammatory bowel disease

The definitive version is available at www.blackwell-synergy.comBackgroundInflammatory bowel disease (IBD) is associated with altered sensory and motor function in the human colon. The aim of the present study was to compare neuromuscular function in normal and IBD-affected colon in vitro, with emphasis on inhibitory enteric nerves, sensory neuropeptides and stimulation of axon collaterals.MethodsStrips of longitudinal and circular muscle were prepared following colectomy from six patients with intestinal carcinoma (mean age 64.2 +/- 4.8 years) and six patients with IBD (Crohn's disease, n = 3; ulcerative colitis, n = 3: mean age 35.8 +/- 5.7 years). Responses were measured to electrical field stimulation, potassium chloride, 1,1-dimethyl-4-phenylpiperazinium iodide, isoprenaline, calcitonin gene-related peptide (CGRP), capsaicin and neurokinin (NK)-1 and -2 receptor subtype-specific agonists, alone or after muscle precontraction.ResultsThe NK-1 and CGRP receptor-mediated relaxation was reduced in the circular (by 44%, P ConclusionsColonic muscle strips from patients with IBD exhibited impaired CGRP and NK-1 receptor-mediated relaxation and NK-2 receptor-mediated contraction. Capsaicin-activated relaxation of colonic smooth muscle is deficient in IBD-affected colon. These results suggest a discrete effect of IBD on sensory-motor coupling and tachykinin-mediated effects on colonic motility.Alicia S Smith and Scott D Smi

Altered nociception, analgesia and aggression in mice lacking the receptor for substance P

The peptide neurotransmitter substance P modulates sensitivity to pain by activating the neurokinin-1 (NK-1) receptor, which is expressed by discrete populations of neurons throughout the central nervous system. Substance P is synthesized by small-diameter sensory 'pain' fibres, and release of the peptide into the dorsal horn of the spinal cord following intense peripheral stimulation promotes central hyperexcitability and increased sensitivity to pain. However, despite the availability of specific NK-1 antagonists, the function of substance P in the perception of pain remains unclear. Here we investigate the effect of disrupting the gene encoding the NK-1 receptor in mice. We found that the mutant mice were healthy and fertile, but the characteristic amplification ('wind up') and intensity coding of nociceptive reflexes was absent. Although substance P did not mediate the signalling of acute pain or hyperalgesia, it was essential for the full development of stress-induced analgesia and for an aggressive response to territorial challenge, demonstrating that the peptide plays an unexpected role in the adaptive response to stress.This work was supported by grants from MRC-ROPA, BBSRC, EC and DGICYT.Peer reviewe

What is different about spinal pain?

<p>Abstract</p> <p>Background</p> <p>The mechanisms subserving deep spinal pain have not been studied as well as those related to the skin and to deep pain in peripheral limb structures. The clinical phenomenology of deep spinal pain presents unique features which call for investigations which can explain these at a mechanistic level.</p> <p>Methods</p> <p>Targeted searches of the literature were conducted and the relevant materials reviewed for applicability to the thesis that deep spinal pain is distinctive from deep pain in the peripheral limb structures. Topics related to the neuroanatomy and neurophysiology of deep spinal pain were organized in a hierarchical format for content review.</p> <p>Results</p> <p>Since the 1980’s the innervation characteristics of the spinal joints and deep muscles have been elucidated. Afferent connections subserving pain have been identified in a distinctive somatotopic organization within the spinal cord whereby afferents from deep spinal tissues terminate primarily in the lateral dorsal horn while those from deep peripheral tissues terminate primarily in the medial dorsal horn. Mechanisms underlying the clinical phenomena of referred pain from the spine, poor localization of spinal pain and chronicity of spine pain have emerged from the literature and are reviewed here, especially emphasizing the somatotopic organization and hyperconvergence of dorsal horn “low back (spinal) neurons”. Taken together, these findings provide preliminary support for the hypothesis that deep spine pain is different from deep pain arising from peripheral limb structures.</p> <p>Conclusions</p> <p>This thesis addressed the question “what is different about spine pain?” Neuroanatomic and neurophysiologic findings from studies in the last twenty years provide preliminary support for the thesis that deep spine pain is different from deep pain arising from peripheral limb structures.</p