Search

Unsuccessful replication attempt of Study 1 from the original paper by Khan & Dhar (2006).

Preregistrations and data related to Luyten et al. 2021. Preprint can be found here: https://www.biorxiv.org/content/10.1101/2020.07.08.193383v2. This paper is now published in BMC Biology: https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-021-00957-x

Visual Abstract, The possible presence of pathological changes in cholinergic synaptic inputs [cholinergic boutons (C-boutons)] is a contentious topic within the ALS field. Conflicting data reported on this issue makes it difficult to assess the roles of these synaptic inputs in ALS. Our objective was to determine whether the reported changes are truly statistically and biologically significant and why replication is problematic. This is an urgent question, as C-boutons are an important regulator of spinal motoneuron excitability, and pathological changes in motoneuron excitability are present throughout disease progression. Using male mice of the SOD1-G93A high-expresser transgenic (G93A) mouse model of ALS, we examined C-boutons on spinal motoneurons. We performed histological analysis at high statistical power, which showed no difference in C-bouton size in G93A versus wild-type motoneurons throughout disease progression. In an attempt to examine the underlying reasons for our failure to replicate reported changes, we performed further histological analyses using several variations on experimental design and data analysis that were reported in the ALS literature. This analysis showed that factors related to experimental design, such as grouping unit, sampling strategy, and blinding status, potentially contribute to the discrepancy in published data on C-bouton size changes. Next, we systematically analyzed the impact of study design variability and potential bias on reported results from experimental and preclinical studies of ALS. Strikingly, we found that practices such as blinding and power analysis are not systematically reported in the ALS field. Protocols to standardize experimental design and minimize bias are thus critical to advancing the ALS field.

Visual Overview, Cocaine- and amphetamine-regulated transcript (CART) is one of the most abundant neuropeptides in vagal afferents, including those involved in regulating feeding. Recent observations indicate that metabolic challenges dramatically alter the neuropeptidergic profile of CART-producing vagal afferents. Here, using confocal microscopy, we reassessed the distribution and regulation of CART(55–102) immunoreactivity in vagal afferents of the male mouse in response to metabolic challenges, including fasting and high-fat-diet feeding. Importantly, the perikarya and axons of vagal C-fibers were labeled using mice expressing channelrodhopsin-2 (ChR2-YFP) in Nav1.8-Cre–expressing neurons. In these mice, approximately 82% of the nodose ganglion neurons were labeled with ChR2-YFP. Furthermore, ChR2-YFP–labeled axons could easily be identified in the dorsovagal complex. CART(55–102) immunoreactivity was observed in 55% of the ChR2-YFP–labeled neurons in the nodose ganglion and 22% of the ChR2-YFP–labeled varicosities within the area postrema of fed, fasted, and obese mice. The distribution of positive profiles was also identical across the full range of CART staining in fed, fasted, and obese mice. In contrast to previous studies, fasting did not induce melanin-concentrating hormone (MCH) immunoreactivity in vagal afferents. Moreover, prepro-MCH mRNA was undetectable in the nodose ganglion of fasted mice. In summary, this study showed that the perikarya and central terminals of vagal afferents are invariably enriched in CART and devoid of MCH.

Better analgesic drugs are desperately needed to help physicians to treat pain. While many preclinical studies support the analgesic effects of α-conopeptides, Vc1.1 and RgIA, the mechanism is controversial., Chronic pain is very difficult to treat. Thus, novel analgesics are a critical area of research. Strong preclinical evidence supports the analgesic effects of α-conopeptides, Vc1.1 and RgIA, which block α9α10 nicotinic acetylcholine receptors (nAChRs). However, the analgesic mechanism is controversial. Some evidence supports the block of α9α10 nAChRs as an analgesic mechanism, while other evidence supports the inhibition of N-type CaV (CaV2.2) current via activation of GABAB receptors. Here, we reassess the effect of Vc1.1 and RgIA on CaV current in rat sensory neurons. Unlike the previous findings, we found highly variable effects among individual sensory neurons, but on average only minimal inhibition induced by Vc1.1, and no significant effect on the current by RgIA. We also investigated the potential involvement of GABAB receptors in the Vc1.1-induced inhibition, and found no correlation between the size of CaV current inhibition induced by baclofen (GABAB agonist) versus that induced by Vc1.1. Thus, GABAB receptors are unlikely to mediate the Vc1.1-induced CaV current inhibition. Based on the present findings, CaV current inhibition in dorsal root ganglia is unlikely to be the predominant mechanism by which either Vc1.1 or RgIA induce analgesia.