Xenopus metamorphosis, developing limb motoneurons must reach their target muscles through the already established larval cholinergic axial neuromuscular system. Here, we demonstrate that at metamorphosis onset, spinal neurons retrogradely labeled from the emerging hindlimbs initially express neither choline acetyltransferase nor vesicular acetylcholine transporter. Nevertheless, they are positive for the motoneuronal transcription factor Islet1/2 and exhibit intrinsic and axial locomotor-driven electrophysiological activity. Moreover, the early appendicular motoneurons activate developing limb muscles via nicotinic antagonist-resistant, glutamate antagonist-sensitive, neuromuscular synapses. Coincidently, the hindlimb muscles transiently express glutamate, but not nicotinic receptors. Subsequently, both pre- and postsynaptic neuromuscular partners switch definitively to typical cholinergic transmitter signaling. Thus, our results demonstrate a novel context-dependent re-specification of neurotransmitter phenotype during neuromuscular system development.In vertebrates, functional motoneurons are defined as differentiated neurons that are connected to a central premotor network and activate peripheral muscle using acetylcholine. Generally, motoneurons and muscles develop simultaneously during embryogenesis. However, during  Islet1 is the first molecular marker of MN differentiation , whereas fore- and hind-limb MNs are located in the brachial and lumbar lateral motor columns (LMC), respectively. In the amphibian tivation . Thus, ativation , when thXenopus initially share the same anatomical and neurochemical environment, and because such features influence neuromuscular development  was next performed on confocal image stacks in which appendicular MNs were identified by retrograde tracer labeling. The fluorescence variation (∆F/F) measured at stages 49–54 showed that the immuno-signal detected in the appendicular motor column was not significantly higher than the background signal level  was performed for ChAT mRNA detection in a larval group ranging from stages 49 to 57. No signal was detected in the appendicular motor column area in stages 49–52 , to become strongly evident from stage 55 onwards  revealed a capability for locomotor-related action potential firing in all recorded MNs , remained strictly coordinated with the ongoing axial pattern  the putative expression and relative distribution of nicotinic and glutamate receptors with fluorescent α-bungarotoxin and the anti-NR2b antibodies, respectively. At stage 52, there was a total lack of labeling for α-bungarotoxin in hindlimb buds . In addiThe first α-bungarotoxin positive signal was detected at stage 55 with characteristic dispersed dots (data not shown), as previously described . By stagd-tubocurarine or CNQX/AP5 cocktail was then bath-applied to test whether these neuromuscular responses were ACh- or glutamate-mediated, respectively. At stages 52–54, the appendicular nerve stimulus-evoked EMG response was not diminished by subsequent d-tubocurarine application, but rather, was increased by 35 ± 6%  but was not significantly affected  in the presence of CNQX/AP5  from stage 55 onwards. This therefore confirms the occurrence of a functional switch in Islet transcription factor, exhibit characteristic MN electrical properties and synaptic influences, are involved in locomotor bouts of activity, and project to limb bud muscles to evoke typical post-junctional responses. Unexpectedly, however, these MNs do not at this premature stage use standard cholinergic neurotransmission to activate their target muscles, but it is not until stage 55 that the limb MNs and their NMJs undergo a simultaneous switch from non-cholinergic to acetylcholine-dependent signaling. Thus our data show for the first time that in metamorphosing Xenopus, MNs that innervate the newly emerging hindlimbs first develop through the employment of a transient, but functional, alternative transmitter mechanism before a conventional and definitive cholinergic phenotype appears.In this study, we report that as early as pre-metamorphosis , de novo appendicular MNs express the Xenopus the ontogeny of appendicular MNs does not obey this well-established developmental pattern, since the present data indicate that before the acquisition of a cholinergic phenotype, these MNs are able to transmit centrally-generated motor commands to the developing limb bud muscles using a different transmitter effector(s). Several lines of evidence indicate that glutamate plays a major role in this precursor signaling at early metamorphic stages. Immunolabeling of stage 52 preparations revealed the presence of the vesicular glutamate transporter VGluT1 in identified (retrogradely-labeled) hindlimb-bud MNs, both in their cell bodies, as has been reported in mammalian CNS neurons  transmitter(s) to another within the same presynaptic neuron and a concomitant modification in associated postsynaptic receptor subtype in order to ensure synapse functionality. This loss/gain in pre-synaptic neurotransmitter/post-synaptic receptor phenotype has been reported to occur in developmental, post-lesional or activity-dependent contexts and is considered to be a major underlying feature of neuronal plasticity . It can d-tubocurarine) might have been due to the low concentrations of glutamate ionotropic receptor antagonists used in our experiments . Animals were maintained at 20–22°C in filtered water aquaria with a 12:12 hr light/dark cycle. Developmental stages were sorted according to external body criteria . After recovering from anesthesia, larvae were kept in a water tank for 24–48 hr to allow tracer migration into MN cell bodies and dendrites. In a series of experiments (n = 4), the hindlimb buds of stages 51–52 larvae were injected and the animals were kept for several days in a separate aquarium until reaching metamorphic climax , University of Iowa, Iowa city, US). Secondary antibodies were donkey anti-goat and anti-rabbit or anti-goat and anti-mouse IgGs coupled to Alexa Fluor 488 and 568 .After MN retrograde labeling, spinal cords were dissected out and fixed in 4% paraformaldehyde (PFA) for 12 hr at 4°C. Preparations were incubated in a 20% [in phosphate-buffered saline (PBS) 0.1%] sucrose solution for 24 hr at 4°C, then embedded in a tissue-tek solution  and frozen at −45°C in isopentane. 40 µm cross-sections were cut using a cryostat . Fluorescence immunohistochemistry was carried out on these spinal cross-sections using the same protocol as described previously . BrieflyFluorescent immunohistochemistry was carried out on entire or 20 µm sliced hindlimb buds from developmental stages 51 to 57 after overnight fixation in PFA 4%. The same labeling protocol was used as described above for spinal cross-sections. Alexa Fluor 488-conjugated α-bungarotoxin  was used to label neuromuscular junctions. The primary antibody, mouse anti-neurofilament associated protein , was used to label nerve branches innervating the limb bud. The primary antibodies, rabbit anti-synaptophysin , goat anti-NMDA subunit one receptor  or rabbit anti-NR2b , and guinea pig or mouse anti-VGluT1 , were used to label synapses, glutamate receptors, and vesicular glutamate transporters, respectively. Note that comparable results were obtained with both anti-VGluT1 antibodies. The bud preparations were then incubated with secondary antibodies donkey anti-mouse, anti-rabbit and anti-goat IgGs coupled to Alexa Fluor 488, 568 and 647 . For microscope imaging whole-mount limb buds were mounted on cavity slides in a homemade medium (see above).Whole-mount preparations and cross-sections labeled with fluorescent material were imaged using an Olympus FV1000 confocal microscope equipped with 488, 543 and 633 nm laser lines. Images were processed using Fiji and Photoshop softwares. Multi-image confocal stacks with 1 μm z-step intervals were generated using a 20x/0.75 oil objective and with 0.3 µm z-step intervals using a 60x/1.4 oil objective. Figure images were obtained by orthogonal projection from multi-image stacks with artificial fluorescent colors using the freeware Fiji.Islet1/2 fluorescence quantifications were performed on original images from 0.3 μm z-step stacks. Fluorescence intensity was measured automatically from 3 ROI in single planes with the same size, defining the slice background, and the axial and appendicular MNs fluorescence signals, respectively. The variation of fluorescence (∆F/F = (F-F0)/F0) was calculated for both axial and appendicular MNs ROI relative to background, the latter being acquired in a ventral spinal region devoid of axial and limb MN cell bodies, yet where cholinergic terminals were present. This calculation was performed on five consecutive confocal planes per slice where the appendicular motor column was identified by retrograde labeling  and where background noise was maximal. Preparations with concomitant ChAT and VAChT fluorescent labeling were combined for statistical quantification  that exhibit about 87% nucleotide identity. This prompted us to generate two ChAT probes. To synthesize these probes, two PCR fragments of 529 (ChAT1) and 512 (ChAT2) bp were amplified from adult X. laevis brain and spinal cord RACE-ready cDNA . Frontal sections (20 µM) of the trunks were cut at −20°C using a cryostat, collected on Superfrost Plus slides , dried at room temperature for 24 hr and stored at −80°C until use.2, 100 mM NaCl, and 0.1% Tween) and transferred to BM Purple (Roche) for colorimetric detection. Finally, they were washed twice in PBS to stop the reaction, and then mounted on gelatin-coated slides in Mowiol. Sections were imaged using a Leica DM5500 B microscope connected to LAS V4.1 software. The specificity of the hybridization procedure was verified by incubating sections with the sense riboprobes with which only background signals typical of this type of chemical reaction could be observed.The in situ hybridization protocol used in the present study was adapted from earlier studies  and cons2, 5% CO2) Ringer solution. The preparation was then placed in a recording chamber and continuously superfused with oxygenated Ringer solution . Spontaneous fictive locomotor episodes were recorded from a caudal ventral root , to impose an elevated intracellular chloride concentration corresponding to that of immature neurons (Xenopus (−] recording solution was replaced by a high [Cl−] version that contained (in mM) 70 K-gluconate, 30 KCl, 10 EGTA, 2 MgCl2, 3 Na2ATP, 0.5 NaGTP, 10 HEPES, pH 7.3. Alexa Fluor 488 (Life Technologies), which was added in the patch pipette to fill the recorded neurons, allowed subsequent verification that recorded cells were indeed RDA-positive . After anesthesia in 0.05% MS-222, the brainstem and spinal cord, including spinal segmental ventral roots, were dissected out in cold oxygenated  was exogenously applied to block nicotinic receptor-type cholinergic synapses. Fictive locomotion sequences were generated either spontaneously or triggered by electrical stimulation of the caudal region of the brainstem (Grass stimulator S88), and the spinal swimming pattern was monitored from a caudal ventral root (between segments 12 and 15) with a suction electrode as described above. EMG activities in rostral myotomes (7–10) and hindlimb buds were recorded simultaneously using pairs of 50 µm insulated wire electrodes connected to a differential AC amplifier (A-M System). Both nerve and EMG activities were digitized at 10 kHz , and displayed and stored on computer for offline analysis with Spike2 software (CED).Semi-intact preparations from stages 52 to 57 larvae were used to simultaneously record EMG activity from axial myotomes and hindlimb bud muscles ; n = 12.Discharge rates in individual nerve and EMG recordings were measured by setting an amplitude threshold to count all impulses in such multi-unit recordings. Firing rates (in spikes/s) were averaged over 10–20 locomotor cycles. Cycle period was taken as the interval between the onsets of two consecutive ventral root bursts. These consecutive burst onsets were used as a trigger for averaging the discharge rates of each EMG channel over cycle duration.Isolated hindlimb-bud preparations with the sciatic and crural nerves still attached ; n = 17 d-tubocurarine or a cocktail of 20 µM CNQX +10 µM AP5 were added to the perfusion solution to block nicotinic or glutamatergic receptors, respectively . Because of the typical difficulty in washing-out these drugs in such experiments, a second antagonist was generally added while the first one was still present in the bath. Control experiments where only one drug was applied showed effects similar to those of combined drug application, and thus data from either approach were pooled in this study. In some experiments on early stages, the normal Ringer solution was replaced by a solution containing half (1 mM) of the normal concentration of MgCl2 in order to unmask any NMDA receptor-mediated component of the EMG’s glutamatergic response. Despite causing a noticeable increase in the control EMG amplitude, such a low Mg2+ solution had no influence on the effects resulting from subsequent antagonist application.30 µM After signal processing in Spike2, electrophysiological data were analyzed using Prism5  and OriginPro8 . Data are shown as means and standard errors of the mean (± SEM), unless stated otherwise. For ChAT and VAChT immunofluorescence signals , differe In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Xenopus" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor.Thank you for submitting your article "Functional limb muscle innervation prior to cholinergic transmitter specification during early metamorphosis in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers found your study on changes in limb motor neuron transmitter phenotype and the composition of receptors at the frog neuromuscular junction of interest and well-executed. The reviewers welcomed your evidence that during metamorphosis when the limbs are forming and becoming innervated, motoneurons initially release glutamate, and then acetylcholine at their synapses on limb musculature. They agree that the manuscript is clearly written and for the most part well-illustrated, especially the use of retrograde labeling to identify and distinguish appendicular and axial motoneurons.eLife's expected timeline for revisions:However, the three reviewers concurred on amending four aspects, all of which would greatly strengthen your study and begin to shed light on mechanism. They expected that these amendments could be accomplished within 1) Demonstration of the presence of any of the known vesicular glutamate transporters in motoneurons at the expected ages.2) Better explanation in the text of the large effect of d-tubocurarine. There is a discrepancy in the reported effects of applying t-tubocurarine at Stage 53, leading to a large decrease in amplitude of EMG activity, and the localization of receptors. Reviewer 2 asks whether you could test the effects of tubocurarine or glutamate antagonists at an earlier stage .3) Inclusion in the Results section of the electrophysiological recordings showing the effects of glutamatergic antagonists. This experiment was alluded to in the Materials and methods section in which a cocktail of CNQX and AP5 was used to block glutamatergic receptors and are critical to the arguments of the paper.4) Better and more complete statistical analyses: Reviewer 3 notes a lack of statistical analysis and an indication of the number of experiments used to generate the quantitative data, a lack of statistical comparison between the fluorescence levels at the different ages, and confusion over standard error of the mean and standard deviation used to illustrate the variability.Reviewer #1:1) Subsection “Locomotor-related activation of hindlimb muscles by non-cholinergic motoneurons”, "is initially largely independent of ACh signaling in…" The amplitude is reduced by more than 50% by d-tubocurarine. How about "is initially only partially suppressed in…."? Could the residual signals be stimulus artifacts? In contrast to Control and Wash, the amplitudes are constant in amplitude. The Materials and methods section describes experiments in which a cocktail of CNQX and AP5 was used to block glutamatergic receptors. These experiments should be illustrated here. They are critical to the story.Related to this point, subsection “Developmental switch in hindlimb neuromuscular transmission”, "indicating the absence of anatomically detectable muscle ACh receptors" would be more appropriate. Figure 4B suggests that they are detectable by physiological / pharmacological methods.Again, related to this point, in the Discussion section, I find "indicative of a complete neurotransmitter phenotype switching…" too great a claim, given that d-tubocurarine substantially reduces the amplitude of the l-HLemg in Figure 4B.2) Discussion section, the authors argue against a muscle spindle innervation role for the limb motoneurons they have studied, on the basis of the sizes of the muscle spindle- and muscle fiber-innervating populations of neurons. However, in the absence of information about the sizes of these populations this is not convincing.3) Quantification is inadequate. The numbers of animals in which observations were made should be provided in each figure legend. Contrary to the assertion in the Transparent Reporting Form, the number of replicates is largely omitted in the Results section and Figure Legends.Reviewer #2:1) Figure 4: It is argued that the switch from glutamatergic to cholinergic transmission occurs around stages 54-55. However, the results presented in Figure 4 clearly show that cholinergic transmission is at place already at stage 53 and possibly even at earlier stages. There is a large decrease in the amplitude of the EMG activity by d-tubocurarine.Could these experiments be done at stage 52? Alternatively, is it experimentally possible to apply glutamatergic antagonists locally on the limb bud?The results of Figure 4 show clearly that the EMG activity at stage 53 is largely mediated by t-tubocurarine-sensitive receptors. This is at odds with the main conclusion of this study.2) Figure 5C,D: The EMG activity recorded at stage 53 is very weak and this may indicate that the neuromuscular junction is not fully developed.The authors should provide electrophysiological traces showing the effects of glutamatergic antagonists. Also, the weak EMG activity recorded at stage 53 might be due to the absence of activation of NMDA receptors. I suggest to repeat these experiments using a Mg-free extracellular solution.3) Overall, it is not clear if limb motor neurons switch the transmitter phenotype or if they use both glutamate and acetylcholine as co-transmitters with a dominance of the one or the other at different developmental stages. In this regard, the authors could test if limb motor neurons co-express transporters for glutamate and acetylcholine in different proportions at different stages.Reviewer #3:The central thesis of this paper is that motoneurons transiently release glutamate instead of acetylcholine at the neuromuscular junction of developing hindlimb muscles at the onset of metamorphosis. The main evidence in support of this conclusion comes from the immunocytochemistry showing that appendicular motoneurons do not express either ChAT or VAChT immunoreactivity and exhibit very little ChAT mRNA at stage 53. Moreover, at stage 52, the developing limb muscle has very low levels of α-bungarotoxin but does express NMDA receptor subunits some of which are co-localized with neuronal synaptophysin .Unfortunately, the physiological experiments do not completely support the anatomy. For example, at stage 53 bath application of d-tubocurarine greatly reduces the amplitude of the rhythmic EMG signals from the limb bud during locomotor episodes despite the apparent absence of acetylcholine receptors in the muscle and cholinergic markers in limb motoneurons. The authors do not explain why the EMG is reduced at this age. Similarly, in the nerve/limb preparation illustrated in Figure 5, bath application of CNQX/AP5 only reduces the stimulus-evoked EMG by 50%. Why is the block incomplete? Again, this is not discussed by the authors. The authors showed only the presence of NMDA receptor subunits in the muscle but did not show the effect of NMDA antagonists alone on the evoked EMG to validate their presence.In both experiments, it would have been useful to combine the glutamatergic and cholinergic antagonists to see if this produced a complete blockade of the muscle electrical activity.Finally, in Figure 5D, the results for control and d-tubocurarine are shown, but it would also be of relevance to show a recording for the glutamate antagonists. Here, the number of experiments is missing, and no p-values are reported.The intracellular recordings of the locomotor activity illustrated in Figure 3E are puzzling. The recordings reveal rhythmic hyperpolarizing potentials and the cells do not fire. Was this true of all of the motoneurons that were recorded? If so, how are the muscles activated if the motoneurons do not spike? For the 11 recorded motoneurons, the authors should indicate from how many preparations they were obtained, and if the motoneurons were only identified by their labeling or whether they tested for an antidromic response. If it is the former, did the authors ever use a second dye applied intracellularly to confirm that the labelled motoneuron was indeed the one that was recorded from.At the earliest stages of limb formation, it was stated that motoneurons were small cells. However, the resistance of the cell shown in Figure 3C was between 3 to 5 MOhm suggesting either a very leaky cell or a large one. What was the input impedance of the other motoneurons?As already alluded to, a major concern is the lack of statistical analysis and an indication of the number of experiments used to generate the quantitative data. For example, in subsection “Delayed cholinergic transmitter phenotype expression in the developing appendicular motor Column” there is no statistical comparison between the fluorescence levels at the different ages nor is the number of experiments shown. Similar problems apply to the quantification in other parts of the paper. The authors often refer to the mean and the SE. Do they mean the standard error of the mean (S.E.M). Why was the standard error of the mean rather than the standard deviation used to illustrate the variability?The quantification of the fluorescence in Figure 2C was confusing. In the text, it states that ChAT and VAChT were not immuno-detected in appendicular MNs until stage 55. The figures in Figure 2A confirm this. However, in the plot of 2C (52-54), which shows the AP/AX fluorescence ratio, the values range from ~0-14% for ChAT. It is difficult to see how these values are generated if AP is zero. The authors should indicate how the ratio was calculated.Also, despite an increase in the ratio at later stages, it is still about 20% of the axial fluorescence (when appendicular MNs are supposed to be bigger). This implies that the majority of appendicular motoneurons are still not cholinergic. It would be helpful to show that once the metamorphosis is complete, this ratio is 100% thereby validating the measure.I found the in-situ hybridization results shown in Figure 2D, difficult to interpret. It is difficult to know the extent of the ChAT ISH labelling in these Figures For example, the extent of the Axial motor column in D 53 looks much more limited than the ChAT labelling in A 53. Furthermore, it is not clear that appendicular MNs at stage 53 are negative. The background is very high. Sections with dextran labelling would have made clear where the motoneurons are located. This figure does not agree with the statement that ISH is more sensitive than immunodetection. How many times was this experiment performed?Xenopus" for further consideration at eLife. Your revised article has been favorably evaluated by Eve Marder (Senior Editor), a Reviewing editor, and three reviewers.Thank you for resubmitting your work entitled "Functional limb muscle innervation prior to cholinergic transmitter specification during early metamorphosis in The manuscript has been improved and reviewers 1 and 2 were satisfied with your amendments, but there are some remaining issues raised by reviewer 3 that remain to be addressed, indicated in the appended reviews. The other reviewers and I in consultation now agree on the need for these revisions:1) The verity of the VGluT1 localization, in synaptic terminals rather than in cell bodies; the predicted restriction of vGluT1 to motor neuron terminals and colocalization with synaptic proteins, and mRNA localization in motor neuron cell bodies; and a comparison to IHC of vGluT2. These aspects were deemed essential to your next revision.The next two sets of comments could be dealt with in the text of the Discussion section:2) incomplete blockade of evoked muscle action by glutamate antagonists, that could be explained by the low concentration of glutamate antagonists used;3) physiology experiments showing that the reduction of the calcium transients generated during fictive swimming by tubocurarine may be due to blockade of GABA-A or glycinergic receptors rather than cholinergic receptors.Reviewer #1:The authors have addressed my concerns satisfactorily. Demonstration of the presence of vGluT1 and the effective use of glutamate receptor antagonists were key. The data in Figure 6B,C are nicely characterized as a "functional switch in hindlimb neuromuscular transmission".Reviewer #2:The authors revised this manuscript according to the criticisms raised by the reviewers. The responses provided reasonable explanations and interpretations.Reviewer #3:In response to the reviewers' concerns the authors have attempted to demonstrate that motoneurons express VGluT1. Unfortunately, I did not find their presentation of the data convincing. For example, VGluT1 expression at stage 52  is widespread throughout the ventral cord and appears, surprisingly, to label cell bodies. This is in contrast to numerous other immunocytochemical studies of VGluT1 labelling in the spinal cord that show the labelling is restricted to synaptic terminals. . The authors should illustrate an image of the whole transverse section of the cord to show how widespread the labelling is. Was this antibody tested or has it been described in other papers to show that it is indeed targeting VGluT1? What experiments did the authors perform to examine the specificity of the antibody? The VGluT1 labelling shown in Figure 5B appears coextensive with the muscle nerve but there is no evidence of the more punctate labelling that characterizes the neuromuscular junction and is shown in Figure 5A (α-bungarotoxin). Furthermore, the authors should show the VGluT1 labelling at stage 57 when glutamatergic transmission is absent. Finally, it was not clear if the authors had tried to label the cells with VGluT2 whose mRNA has been reported in some studies to be expressed in the neonatal mammalian motoneurons.A more convincing approach to demonstrate the presence of a vesicular protein in motoneurons would be to show that it co-localizes with a synaptic protein – such as synaptophysin at the neuromuscular junction or to demonstrate the presence of the mRNA in motoneuronal cell bodies. Essential revisions:1) Demonstration of the presence of any of the known vesicular glutamate transporters in motoneurons at the expected ages.New experiments on the immuno-detection of VGluT1 have demonstrated the presence of the glutamate vesicular transporter in early stage tadpoles, both at the level of motor axons in the developing hindlimbs  and the limb motoneuron cell bodies themselves .2) Better explanation in the text of the large effect of d-tubocurarine. There is a discrepancy in the reported effects of applying t-tubocurarine at Stage 53, leading to a large decrease in amplitude of EMG activity, and the localization of receptors. Reviewer 2 asks whether you could test the effects of tubocurarine or glutamate antagonists at an earlier stage .d-tubocurarine could be attributable to an action at either central or peripheral levels, or both. In comparing the results of the experiments illustrated in Figure 4 and old Figure 5, it seemed reasonable to us to interpret the EMG amplitude changes seen under d-tubocurarine at stage 53 as being centrally-mediated in Figure 4 and peripherally-mediated in old Figure 5. Manifestly, however, this was not obvious to the reader. We now also provide new data  showing that cholinergic synapses are present on limb motoneuron cell bodies, that they play an important role in the excitation of these motoneurons during centrally-generated swimming and are directly affected by nicotinic antagonist perfusion. This is demonstrated by new calcium imaging experiments that show a strong reduction in the calcium signal in limb motoneurons during fictive swimming in the presence of d-tubocurarine. These supplementary results, which are briefly described in subsection “Locomotor-related activation of hindlimb muscles by non-cholinergic motoneurons”, thus strongly support the conclusion that d-tubocurarine was indeed having a major central effect on limb motoneuron activation in the experiments of Figure 4B .Evidently there was a general misinterpretation of the results illustrated in Figure 4 and original Figure 5, and we thank the three referees for having brought this lack of clarity to our attention. In old Figure 5 , we used isolated hindlimb preparations to stimulate directly the limb motor nerve, and therefore the antagonists’ effects on the EMG responses could be concluded to be exclusively peripheral, at the neuromuscular junction. In Figure 4, in contrast, the experiments were performed on semi-intact preparations where spontaneously-active spinal CPG circuitry was still present and able to drive motoneurons and EMG activity in both the axial and limb muscles. In this case, any observed effects of 3) Inclusion in the Results section of the electrophysiological recordings showing the effects of glutamatergic antagonists. This experiment was alluded to in the Materials and methods section in which a cocktail of CNQX and AP5 was used to block glutamatergic receptors and are critical to the arguments of the paper.non-cholinergic nature of the transmitter phenotype present at early developmental stages. However, we agree that illustrating the parallel effects of glutamate antagonists does strengthen the phenotype switching message of the article.These data are now illustrated in new Figure 6. We initially believed that illustrating the cholinergic drug effects was important for emphasizing more the 4) Better and more complete statistical analyses: Reviewer 3 notes a lack of statistical analysis and an indication of the number of experiments used to generate the quantitative data, a lack of statistical comparison between the fluorescence levels at the different ages, and confusion over standard error of the mean and standard deviation used to illustrate the variability.The lack of numbers of experiments in the original manuscript was due to an unfortunate editing error for which we apologize. This information is now reestablished, where appropriate, in the text and/or figure legends. The lack of statistical comparison pointed out by reviewer 3 relates specifically to the changes in ChAT and VAChT fluorescence levels found in limb motoneurons during development . Although we originally performed such a comparison, we didn't report it because of uncertainty about its validity due to prepto-prep differences, for example, in the extent of retrograde label uptake, fluorescence intensities, and tissue penetration by both primary and secondary antibodies. However, as requested by the reviewer, this comparative analysis is now provided in Figure 2.We have followed conventional procedure in providing means ± SEM in the text. In original Figure 2C, individual values (circled dots) were presented for single experiments in each age group, allowing their dispersion to be visualized, with the error bars representing the standard error of the overall mean (black dots) of these values per group. Note that this analysis has been redone according to reviewer 3’s remarks and that we have changed the data display to improve clarity.Reviewer #1:1) Subsection “Locomotor-related activation of hindlimb muscles by non-cholinergic motoneurons”, "is initially largely independent of ACh signaling in…" The amplitude is reduced by more than 50% by d-tubocurarine. How about "is initially only partially suppressed in…."? Could the residual signals be stimulus artifacts? In contrast to Control and Wash, the amplitudes are constant in amplitude. The Materials and methods section describes experiments in which a cocktail of CNQX and AP5 was used to block glutamatergic receptors. These experiments should be illustrated here. They are critical to the story.This part of the Results section has been rewritten to incorporate new immunohistochemical and electrophysiological data  which indicate that the reduction in limb muscle EMG under d-tubocurarine in pre-stage 55 preparations was most likely due to a central nervous effect on motoneuron activation rather than a peripheral effect on the neuromuscular junction. Note also that the activity recorded in Figure 4 is spontaneous fictive swimming without involving any electrical stimulation (and thus the possibility of stimulus artifacts).Hindlimb EMG responses to motor nerve electrical stimulation in the presence of glutamatergic antagonists are now illustrated in new Figure 6 for both early and late developmental stages, as requested by the three reviewers and the corresponding text have been modified .Related to this point, subsection “Developmental switch in hindlimb neuromuscular transmission”, "indicating the absence of anatomically detectable muscle ACh receptors" would be more appropriate. Figure 4B suggests that they are detectable by physiological / pharmacological methods.Again, related to this point, in the Discussion section, I find "indicative of a complete neurotransmitter phenotype switching…" too great a claim, given that d-tubocurarine substantially reduces the amplitude of the l-HLemg in Figure 4B.In order to be more circumspect, we have rewritten as “indicating an absence of muscle ACh receptors…” subsection “Developmental switch in hindlimb neuromuscular transmission”.For the same reason, “indicative” has been replaced by “consistent with” in the Discussion section.We agree that Figure 4B could suggest ACh to be involved in the neuromuscular transmission at stage 53. However, as stated in our response to the reviewer’s preceding comment, another explanation is that the observed effect resulted from the d-tubocurarine acting centrally on limb motoneurons during fictive axial swimming . Specifically, these new data show the presence of cholinergic synapses in the vicinity of limb MN cell bodies and a suppressive effect of the nicotinic antagonist on the calcium dynamics of limb MNs during fictive swimming. Taken together with our other results, therefore, these new data add strong support to the conclusion of a complete switch in the neurotransmitter phenotype of limb motoneurons during early metamorphosis.2) Discussion section, the authors argue against a muscle spindle innervation role for the limb motoneurons they have studied, on the basis of the sizes of the muscle spindle- and muscle fiber-innervating populations of neurons. However, in the absence of information about the sizes of these populations this is not convincing.in situ hybridization; Figure 3—figure supplement 1) have revealed the existence of a relatively large population of putative appendicular MNs, at least the greater proportion of which was identified (by back-filling) as projecting into the limb bud. Thus, it seems reasonable to conclude that this extensive population of non-cholinergic limb MNs does not correspond to a possible discrete fusimotor population. The text has been rewritten (Discussion section) to try and clarify this point.A specific fusimotor innervation has not previously been described in frogs, other than by Fujitsuka et al., (1987) who reported the existence of a small population of fusimotor MNs innervating exclusively muscle spindles, and in only one muscle (the semitendinosus). Although not quantified, our data on Islet immunoreactivity (and now from 3) Quantification is inadequate. The numbers of animals in which observations were made should be provided in each figure legend. Contrary to the assertion in the Transparent Reporting Form, the number of replicates is largely omitted in the Results section and Figure Legends.We apologize for this omission, which has been corrected. All numbers of animals and cells are now provided where appropriate in the text and/or figure legends.Reviewer #2:1) Figure 4: It is argued that the switch from glutamatergic to cholinergic transmission occurs around stages 54-55. However, the results presented in Figure 4 clearly show that cholinergic transmission is at place already at stage 53 and possibly even at earlier stages. There is a large decrease in the amplitude of the EMG activity by d-tubocurarine.Could these experiments be done at stage 52? Alternatively, is it experimentally possible to apply glutamatergic antagonists locally on the limb bud?The results of Figure 4 show clearly that the EMG activity at stage 53 is largely mediated by t-tubocurarine-sensitive receptors. This is at odds with the main conclusion of this study.This important point was also raised by reviewer 1. Our response is the same and is as follows: We agree that Figure 4B could suggest that ACh is involved in transmission at the NMJ of the de novo limb muscles at stage 53. However, another (and we think more likely) explanation is that the decreased EMG response to d-tubocurarine is due to the drug acting centrally in diminishing the premotor activation of limb motoneurons during fictive axial swimming in these semi-isolated  preparations. In support of this idea, we provide new immuno-anatomical labeling evidence for the presence of VAChT co-localized with synapsin around limb motoneuron cell bodies, indicating the presence of central cholinergic synapses on these neurons. That these synapses are involved in driving the limb MNs was confirmed with calcium imaging experiments on isolated CNS preparations demonstrating that their activation is substantially decreased during fictive swimming in the presence of d-tubocurarine. These additional results, which are illustrated in Figure 4—figure supplement 1 and briefly described in subsection “Locomotor-related activation of hindlimb muscles by non-cholinergic motoneurons” are therefore consistent with our study’s main conclusion that a switch in neurotransmitter phenotype of limb motoneurons occurs during early metamorphosis.Finally, applying glutamatergic antagonists to the limb buds is certainly a potentially instructive approach and one that we had already attempted. Unfortunately, due the extremely small size of the limb buds in early stage animals (50-53), injecting drugs without destroying bud neuromuscular tissue and while maintaining a muscle recording electrode in place was found to be technically impossible.2) Figure 5C,D: The EMG activity recorded at stage 53 is very weak and this may indicate that the neuromuscular junction is not fully developed.The authors should provide electrophysiological traces showing the effects of glutamatergic antagonists. Also, the weak EMG activity recorded at stage 53 might be due to the absence of activation of NMDA receptors. I suggest to repeat these experiments using a Mg-free extracellular solution.The effects of glutamate antagonists on EMG responses to nerve stimulation are now shown in new Figure 6. A series of experiments was also done in saline containing half the normal MgCl2 concentration. As implied by the reviewer, the EMG response at stage 53 was increased  is now taken from these experiments), but the effect of the glutamatergic antagonists  remained similar to that under normal saline.3) Overall, it is not clear if limb motor neurons switch the transmitter phenotype or if they use both glutamate and acetylcholine as co-transmitters with a dominance of the one or the other at different developmental stages. In this regard, the authors could test if limb motor neurons co-express transporters for glutamate and acetylcholine in different proportions at different stages.The expression of the vesicular transporter for glutamate VGluT1 in motor axons at early stages is now illustrated for a stage 53 tadpole in new Figure 5C. In addition, we provide as Figure 5—figure supplement 1 an illustration of VGluT1 immuno-detection in limb motoneuron cell bodies at stage 52, when ChAT and VAChT are not expressed. However, the expression of this transporter does not seem to persist at later stages, thus paralleling the lack of expression of NR1 and NR2b receptor subunits in the limb muscles.Reviewer #3:The central thesis of this paper is that motoneurons transiently release glutamate instead of acetylcholine at the neuromuscular junction of developing hindlimb muscles at the onset of metamorphosis. The main evidence in support of this conclusion comes from the immunocytochemistry showing that appendicular motoneurons do not express either ChAT or VAChT immunoreactivity and exhibit very little ChAT mRNA at stage 53. Moreover, at stage 52, the developing limb muscle has very low levels of α-bungarotoxin but does express NMDA receptor subunits some of which are co-localized with neuronal synaptophysin .Unfortunately, the physiological experiments do not completely support the anatomy. For example, at stage 53 bath application of d-tubocurarine greatly reduces the amplitude of the rhythmic EMG signals from the limb bud during locomotor episodes despite the apparent absence of acetylcholine receptors in the muscle and cholinergic markers in limb motoneurons. The authors do not explain why the EMG is reduced at this age. Similarly, in the nerve/limb preparation illustrated in Figure 5, bath application of CNQX/AP5 only reduces the stimulus-evoked EMG by 50%. Why is the block incomplete? Again, this is not discussed by the authors. The authors showed only the presence of NMDA receptor subunits in the muscle but did not show the effect of NMDA antagonists alone on the evoked EMG to validate their presence.As explained in our responses to similar comments made by reviewers 1 and 2, the experiments in Figure 4 were performed on semi-isolated CNS/muscle preparations where EMG activity in both the axial and limb muscles was driven spontaneously by the spinal locomotor network. We now provide new anatomical and calcium imaging evidence  that cholinergic synapses are present on limb motoneuron cell bodies and are substantially involved in the excitation of these cells during centrally-generated swim episodes. Thus, it is likely that the diminution of the limb EMG strength evident in Figure 4B resulted from a direct central effect of dtubocurarine on limb motoneuron excitation rather than at the peripheral neuromuscular junction.Xenopus axial motoneurons in culture; e.g., work from N. Spitzer’s group). In addition, combining antagonists  never led to a full blockade of stimuli-evoked EMG responses. We chose not to report these additional experiments in the revised manuscript to avoid over-complicating the main message and especially since it seems more generally that glutamate antagonists are unable to completely block glutamate-mediated transmission in amphibians.On the other hand, the use of glutamatergic antagonists on the semi-intact preparation totally abolishes fictive swimming and so an equivalent EMG analysis could not be done with these antagonists. However, both cholinergic and glutamatergic sets of antagonists could be used on the isolated motor nerve-hindlimb preparation with nerve stimulation . The lack of a complete block by glutamatergic antagonists at stage 53  has several possible explanations. The first and simplest is that glutamate antagonists are not fully effective in the amphibian  and cause a partial blockade only, as already reported . Another explanation is that glutamate is not the only neurotransmitter used at the limb neuromuscular junctions in these early stages. In relation to this possibility, we did an additional series of experiments using other neurotransmission blockers, but no clear results emerged .The intracellular recordings of the locomotor activity illustrated in Figure 3E are puzzling. The recordings reveal rhythmic hyperpolarizing potentials and the cells do not fire. Was this true of all of the motoneurons that were recorded? If so, how are the muscles activated if the motoneurons do not spike? For the 11 recorded motoneurons, the authors should indicate from how many preparations they were obtained, and if the motoneurons were only identified by their labeling or whether they tested for an antidromic response. If it is the former, did the authors ever use a second dye applied intracellularly to confirm that the labelled motoneuron was indeed the one that was recorded from.At the earliest stages of limb formation, it was stated that motoneurons were small cells. However, the resistance of the cell shown in Figure 3C was between 3 to 5 MOhm suggesting either a very leaky cell or a large one. What was the input impedance of the other motoneurons?The absence of firing in our recorded motoneurons was due to the internal solution that we generally used in our patch-clamp electrodes. We have performed a new set of experiments in which the patch pipette solution was changed for a high chloride solution, as usually used for recording immature motoneurons . In this latter condition, neurons were able to fire action potentials spontaneously or in response either to current injection or spontaneous CPG activity. These experiments are illustrated in Figure 3C,D and F, and both the Results section and Materials and methods section have been updated accordingly.Recorded motoneurons were indeed identified according to their labeling from hindlimb muscles, although fluorescent dye was also sometimes added in the patch pipette for later verification. This is now stated in the text and illustrated in Figure 3B. The number of preparations in each case is indicated in the Materials and methods section.Early stage appendicular motoneurons have somata of 15-20 µm and were classified as 'small' neurons compared to the large axial motoneuron cell bodies (that are about three-fold larger than those of limb motoneurons). We are fairly confident that the intracellular recordings reported in this study were not ‘leaky’ , but since we have no comparison with other studies in this developmental range, we cannot draw any conclusions about the significance of the resistance values observed.As already alluded to, a major concern is the lack of statistical analysis and an indication of the number of experiments used to generate the quantitative data. For example, in subsection “Delayed cholinergic transmitter phenotype expression in the developing appendicular motor Column” there is no statistical comparison between the fluorescence levels at the different ages nor is the number of experiments shown. Similar problems apply to the quantification in other parts of the paper. The authors often refer to the mean and the SE. Do they mean the standard error of the mean (S.E.M). Why was the standard error of the mean rather than the standard deviation used to illustrate the variability?A statistical comparison was not reported for the fluorescence levels because we doubted the validity of this type of analysis. However, we have changed the way the fluorescence data are displayed, and statistics are also given. Note that this analysis has been redone according to the following comment.The quantification of the fluorescence in Figure 2C was confusing. In the text, it states that ChAT and VAChT were not immuno-detected in appendicular MNs until stage 55. The figures in Figure 2A confirm this. However, in the plot of 2C (52-54), which shows the AP/AX fluorescence ratio, the values range from ~0-14% for ChAT. It is difficult to see how these values are generated if AP is zero. The authors should indicate how the ratio was calculated.Cholinergic terminals are widely present in the spinal cord, including around limb motoneuron somata , which can sometimes affect the fluorescence ratio. We have redone the analysis in choosing as background a region without motoneurons but enriched in cholinergic terminals. This is now stated in the Materials and methods section and we are grateful to the reviewer for pointing out this analytical bias. The results of this analysis are presented differently from the original Figure 3C: we now show ∆F/F for axial and appendicular motoneurons separately, which also allowed reporting the statistics.Also, despite an increase in the ratio at later stages, it is still about 20% of the axial fluorescence (when appendicular MNs are supposed to be bigger). This implies that the majority of appendicular motoneurons are still not cholinergic. It would be helpful to show that once the metamorphosis is complete, this ratio is 100% thereby validating the measure.Our evidence indicates that all limb motoneurons become cholinergic from stage 55 onwards. However, axial motoneurons always displayed much more fluorescence than limb motoneurons, and this is even more evident in the new data representation in Figure 2C. Even in juvenile adults, i.e. after metamorphosis completion, the ratio remains largely in favor of axial motoneurons. Presumably this indicates a higher concentration of cholinergic proteins in axial motoneurons, but we do not know why this is so.I found the in-situ hybridization results shown in Figure 2D, difficult to interpret. It is difficult to know the extent of the ChAT ISH labelling in these Figures For example, the extent of the Axial motor column in D 53 looks much more limited than the ChAT labelling in A 53. Furthermore, it is not clear that appendicular MNs at stage 53 are negative. The background is very high. Sections with dextran labelling would have made clear where the motoneurons are located. This figure does not agree with the statement that ISH is more sensitive than immunodetection. How many times was this experiment performed?Islet1 in situ hybridization has been performed on the same preparations as ChAT hybridization. We now also illustrate the results for Islet1  in order to compare the location and extent of the appendicular motor column at key developmental stages. Whereas limb motoneurons were always Islet1 mRNA-positive, ChAT mRNAs were never detected at early stages . New images are displayed in which the axial motor column is clearly visible in all hybridizations, and the number of experiments has been added in the text (subsection “Delayed cholinergic transmitter phenotype expression in the developing appendicular motor column”).mRNAs were detected with in situ hybridization before their translation into proteins. Thus, at pivotal stages 53-54, a light ChAT mRNA labeling could be observed in some preparations whereas ChAT proteins were never immuno-detected. This again is consistent with a delayed acquisition of the cholinergic phenotype by limb motoneurons.The manuscript has been improved and reviewers 1 and 2 were satisfied with your amendments, but there are some remaining issues raised by reviewer 3 that remain to be addressed, indicated in the appended reviews. The other reviewers and I in consultation now agree on the need for these revisions:1) The verity of the VGluT1 localization, in synaptic terminals rather than in cell bodies; the predicted restriction of vGluT1 to motor neuron terminals and colocalization with synaptic proteins, and mRNA localization in motor neuron cell bodies; and a comparison to IHC of vGluT2. These aspects were deemed essential to your next revision.non-glutamatergic neurons and accordingly, a strong concentration of VGluT1 immunoreactivity was detected in the vicinity of the latters' cell bodies, but which themselves unsurprisingly were nonreactive. In contrast, other studies on glutamatergic signaling in various brain and spinal structures  have reported the expression of VGluT1 both within the somata of glutamatergic neurons, as well as along their axons. Although these studies were on rodents, and we have not found previous reports of similar findings in amphibians, the results do provide a clear precedence for our observation of cell body and axonal VGluT1 expression in immature Xenopus limb motoneurons.A major remaining concern of reviewer 3 was our finding of VGluT1 localization in the immature limb motoneuron cell bodies, where he/she argued  that VGluT1 should be found exclusively in axon terminals, in the vicinity of synaptic contacts. However, we respectfully point out that there is evidence to indicate otherwise. Most previous studies on the subject (including the two reports cited by the reviewer) investigated glutamatergic projections onto Nevertheless, we performed a new set of immunolabeling experiments using a different VGluT1directed antibody . Here again, we obtained a very similar labeling in early limb motoneuron cell bodies  as with the guinea pig antibody. Additionally, VGluT1 expression was found along MN axons, as well as co-localized with synaptophysin and NR1 immuno-labeling  as would be expected with a functional glutamatergic NMJ. As also requested by the reviewer, we now illustrate that VGluT1 labeling is lost in stages older than 54 , when glutamatergic signaling disappears.Unfortunately, after many attempts with various protocols  we failed to produce VGluT hybridization, which if successful would have obviously reinforced our immunohistological observations. However, we believe that (1), finding very similar results with VGluT1directed antibodies originating from two different mammalian sources, (2), the general endorsement from the literature, and (3), the combination of the experimental data provided by our present study support the conclusion that before becoming cholinergic, de novo limb motoneurons at premetamorphosis initially use glutamate as a major transmitter at the NMJ.Xenopus laevis spinal cord. Although our experiments using mammalian VGluT1 antibodies gave positive results, this of course doesn't mean that the Xenopus and guinea pig or mouse VGluT1 isoforms are identical. We also tested for VGluT2 immunolabeling  in spinal cord cross sections and this failed completely. In this context, however, it is relevant that according to a phylogenetic analysis by Villar-Cervino et al., (2010), vesicular glutamate transporters in Xenopus are thought to exist as two related subtypes (a and b) of VGluT1, rather than as homologues of the distinct VGluT1 and 2 isoforms. In any case, we should emphasize that the aim of our VGluT immunolabeling experiments was to seek supporting evidence for (or against) glutamatergic synaptic signaling playing a role in the early developing limb system, rather than attempting to identify the specific transporter isoforms that might be involved.The impetus for our VGluT1 immunolabeling experiments was based on a previous report  demonstrating the transporter's presence in the early developing The next two sets of comments could be dealt with in the text of the Discussion section:2) incomplete blockade of evoked muscle action by glutamate antagonists, that could be explained by the low concentration of glutamate antagonists used;A new paragraph has been added to the Discussion section where we now address the incomplete blockade of evoked EMG responses by glutamatergic antagonists. We have also rewritten much of the Discussion section’s first two paragraphs in order to avoid implying that glutamate is alone responsible for neurotransmission at the early limb NMJ.3) physiology experiments showing that the reduction of the calcium transients generated during fictive swimming by tubocurarine may be due to blockade of GABA-A or glycinergic receptors rather than cholinergic receptors.This interesting and plausible possibility is now mentioned in the Results section. Indeed, the possible contribution of a d-tubocurarine-sensitive, GABA-A receptor-mediated drive to limb motoneurons reinforces our hypothesis that in spontaneously active semi-intact preparations, d-tubocurarine causes a central blockade of motoneuron activation, rather than acting peripherally at the limb NMJ.Reviewer #3:In response to the reviewers' concerns the authors have attempted to demonstrate that motoneurons express VGluT1. Unfortunately, I did not find their presentation of the data convincing. For example, VGluT1 expression at stage 52  is widespread throughout the ventral cord and appears, surprisingly, to label cell bodies. This is in contrast to numerous other immunocytochemical studies of VGluT1 labelling in the spinal cord that show the labelling is restricted to synaptic terminals. . The authors should illustrate an image of the whole transverse section of the cord to show how widespread the labelling is. Was this antibody tested or has it been described in other papers to show that it is indeed targeting VGluT1? What experiments did the authors perform to examine the specificity of the antibody? The VGluT1 labelling shown in Figure 5B appears coextensive with the muscle nerve but there is no evidence of the more punctate labelling that characterizes the neuromuscular junction and is shown in Figure 5A (α-bungarotoxin). Furthermore, the authors should show the VGluT1 labelling at stage 57 when glutamatergic transmission is absent. Finally, it was not clear if the authors had tried to label the cells with VGluT2 whose mRNA has been reported in some studies to be expressed in the neonatal mammalian motoneurons.A more convincing approach to demonstrate the presence of a vesicular protein in motoneurons would be to show that it co-localizes with a synaptic protein – such as synaptophysin at the neuromuscular junction or to demonstrate the presence of the mRNA in motoneuronal cell bodies.Xenopus limb motoneurons finds strong echoes in the literature. Similarly, the expression of VGluT1 along glutamatergic neuron axons was also reported in these studies, which also corresponds to our findings in immature limb motoneuron axons.As detailed in our response to editorial comment 1 above, our observation of a diffuse VGluT1 immuno-reactivity in the somata of early The new series of VGluT1 immuno-detection experiments using a different antibody (generated from mice) provided a test for antibody specificity. The finding of VGluT1 expression in MN somata was the same as with the original guinea pig antibody, and these data are now illustrated in new Figure 5—figure supplement 1A. In addition, combined VGluT1, synaptophysin and NR1 immuno-detection in early hindlimbs showed co-localized labeling, consistent with functional neuromuscular synapses . Finally, we provide new images illustrating the parallel loss of VGluT1 and NR1 immuno-detection in hindlimbs in stages older than 54 .Xenopus, and what is usually termed VGluT2 is in fact a subtype of the VGluT1 isoform due to the tetraploid nature of the animal. Contrary to what is observed in other species, the amino acid sequences differ very little between the two resulting proteins, thus corresponding to VGluT1a and VGluT1b. Although no distinction could be made in our experiments between these two VGluT1 subtypes, this would explain our failure to see any VGluT2 labeling using a mammalian antibody.According to a phylogenetic analysis by Villar-Cervino et al., (2010) a homologue of the mammalian or fish VGluT2 gene doesn't not exist in Frustratingly, our repeated attempts (and with different protocols) failed to achieve VGluT hybridization. However, as stated in our response to editorial comment 1, we believe that the combination of our existing immunolabeling and electrophysiological data provide convincing evidence that pre-metamorphic limb motoneurons initially use glutamate as a neurotransmitter before becoming cholinergic.


Objective: Craving for substance abuse is a usual and complicated problem in patients, with opioid addiction who are in opioid detoxifying process. Craving has been added as one of the diagnostic criteria of substance use disorders in DSM-5. The present trial aimed at comparing the effects of celecoxib versus ibuprofen in reducing pain and decreasing the desire to use opiates in patients undergoing opiate detoxification (n = 32). Method: A total of 32 patients (both inpatients and outpatients), who were undergoing opiate detoxification procedure and met the inclusion criteria entered this 4- week study. Participants who suffered from pain due to opiate withdrawal were randomized into 2 groups: Group 1 received celecoxib 200 milligrams once per day and group 2 received ibuprofen 400 milligrams 4 times per day. Self-reported Desire for Drug Questionnaire (DDQ) and 0-10 numeric pain scale were used at baseline and at the end of the study to evaluate changes in opiate craving and pain, respectively. Data analysis was done by SPSS-21 statistical software.Results: In this study, 16 patients received celecoxib 200 milligrams once daily, and 16 received ibuprofen 400 milligrams 4 times daily. After 4 weeks of treatment with both ibuprofen and celecoxib, the results revealed that celecoxib and ibuprofen equally reduced the pain symptoms. After 4 weeks of treatment, with either ibuprofen or celecoxib, significant improvement was observed in decreasing the craving in the celecoxib group, but not in the ibuprofen group.Conclusion: The study revealed a significant difference between the celecoxib and ibuprofen group in reducing craving in patients with opiate craving after 4 weeks of treatment. However there were no significant differences between these two groups in reducing pain. Craving is very common in patients who undergo opiate detoxifying procedure. Although drug craving has been defined in numerous ways, it has generally been regarded as a compelling urge to use a substance . CravingSeveral mechanisms are involved in drug craving, some of which are as follow: Dopamine system , decisioResearchers have long posited a relationship between craving and addiction . AbstineNon-steroidal anti-inflammatory drugs are inhibitors of the cyclooxygenase enzyme family, which catalyzes the metabolism of arachidonic acid to prostaglandins, prostacyclin, and thromboxane. The cyclooxygenase-1 isoform is constitutively expressed in most tissues. The cyclooxygenase-2 is usually specific to inflamed tissue .Celecoxib is a COX-2 inhibitor that has been used in many psychiatric problems such as major depression by inhibiting prostaglandin E2, which may be raised in MDD  and reduIn our practice, we noted that many psychiatrists believe that celecoxib causes more craving inhibitory effect when compared with other nonsteroidal anti-inflammatory drugs (NSAIDs)and when being used as an analgesic in patients who have craving during their opiate cessation procedure. The researches reflect the action on celecoxib on some parts of the brain involving craving . To our The present study tested the theory that celecoxib would differ from ibuprofen in measuring the desire to use opioids because of its positive effects on brain, and also it was expected to lower the pain in the 2 groups of the study .In summary, this study aimed at testing whether the inhibition of craving effect, assessed terms of increased desire to use opioid for their rewarding effects, would be greater in the celecoxib group than the ibuprofen group, following administration of the 4-week therapy using both medicines.ParticipantsIn this study, 32 opiate addicts, who met DSM-5 criteria for opioid use disorder, participated. Patients were under a detoxification protocol at addiction clinics in Tehran, Iran. They were randomly allocated into 2 groups and they received a 30-day celecoxib treatment or a 30-day ibuprofen treatment for their pain during their detoxifying period. The inclusion criteria included patients aged 18 and 65 who were admitted to Roozbeh hospital clinic and a private clinic to receive opioid cessation regimens.The exclusion criteria included pregnancy and lactation, suffering from severe medical conditions , or neurological illnesses including epilepsy and Parkinsonism, having received NSAIDs within the week prior to the initiation of the study as well as having intellectual disability.There were no significant differences in the demographic variables or drug history between the participants of the 2 groups. All participants were identical in environment and lifestyle during the trial; for instance, they were received similar diet and drug regimen and the same psychiatric counseling during their therapy. Moreover, they were asked not to change their usual lifestyle during the trial. The detoxifying procedure was done by standard psychiatric counselling of psychiatrists and psychologists. No other routine detoxifying medications (such as methadone) were used.ProcedureEach patient participating in this study was assessed for pain and craving for opiates at baseline. The patients were assigned to receive either celecoxib or ibuprofen randomly for 4 weeks. Patients in celecoxib group received celecoxib capsule 200 milligrams orally once daily at 12 PM. Ibuprofen was administered in a soft gel form and in an equal dose of celecoxib, which is 1600 milligrams in 4 divided doses at 08:00 AM, 02:00 PM, 08:00 PM and 12:00 midnight . The dosA psychiatry resident randomly assigned the patients to receive treatment with either celecoxib or ibuprofen under single-blind conditions and allocated them to parallel groups, which was determined by simple randomization. He kept the randomization list secure during the study.After being provided with a full explanation of the protocol design, all patients provided written informed consent. This trial was approved by the Ethics Committee of the Faculty of Pharmacy at Tehran University of Medical Sciences and registered in the Iranian Registry of Clinical Trials . All phases of this study were performed according to the declaration of Helsinki on ethical principles for medical research involving human subjects.Questionnaires and MeasuresThe 14-item Desire for Drug Questionnaire (DDQ) is an effective tool for subjective assessment, showing how strong the desire is to use opiates  . It is athis study to assess the intensity of the patients’ pain. It is a numeric scale, ranging from 0-10, with zero indicating no pain and 10 the worst possible pain .Prior to the beginning of the study, sample size was calculated to detect a 20% difference in improvement between celecoxib and ibuprofen. Under the assumption of a significant level of 0.05 with a power of 0.80, a minimal sample size of 32 with 16 cases in each group was determined. Estimating a dropout rate of 20%, we decided to recruit 20 participants for each group.Data AnalysisData analysis was done by SPSS-21 statistical software. The changes in pain and craving were detected at Weeks 0 and 4 between the 2 groups. Statistical significance was set at p < 0.05.Demographic CharacteristicsDemographic characteristics of the patients in both groups are demonstrated in The 0-10 numeric pain scale  was also used inParticipants who were assigned into the 2 groups did not differ in their gender, age, marital, job or educational status . AnalysiPain SeverityAt first for analyzing the scores the outlier test was passed. To analyze pain severity, independent samples Mann-Whitney U test, a non-parametric test, was used because the shapiro-wilk test showed a significant difference (p<0.05). The result of pain score is presented in Inhibitory Effects on Craving=0.004). The results did not indicate a significant difference at the pretest point for craving symptoms. So to compare the 2 groups of treatment, analysis of covariance (ANCOVA) was used with pretest score as the covariant. The ANCOVA results and the means and standard deviation of the outcome measures at the pretest and posttest are presented in  = 12.94, p = 0.001)  was used to determine the equivalences of the treatment conditions on outcome measures at the pretest point and the pre-hypothesis tests were done before analysis. The general linear model test also was not significantly different at the pretest  has proposed that the biological basis for drug ‘craving’ reflects 2 independent processes: one in which the primary objective is to alleviate an aversive state , and the other in which appetitive motivational processes are involved .Frolov’s trial shows that celecoxib as a COX-2 Inhibitor, can act in several parts of the brain and in those parts responsible for craving .In another study, Dembo et al. found that celecoxib penetration into the CNS is indicated by the CSF plasma concentration ratio .Ciceri et al. showed that celecoxib reduced the elevated PGE2 levels in the brain of kainate-treated rats and inhibited induced COX activity, demonstrating the ability of this compound to act on COX-2 in CNS .Beiche et al. found that mRNA of both COX isoforms is expressed continuously in the spinal cord with COX-2 as the predominant isoform .Celecoxib administration produces a decrease in the subjective desire to use opiates for their pleasant effects. There was no overall significant difference on this measure between the 2 groups at baseline so these results reflected no pre-existing differences prior to drug administration day. However, the pattern of results obtained revealed significant differences between the ibuprofen and celecoxib group on craving. However, no significant difference was obtained between the 2 groups of treatment on reducing pain.The author suggests considering high risk patients such as patients who have GI diseases, cardiac diseases and etc., before starting celecoxib or ibuprofen for them. Furthermore, regular monitoring of the patients after initiating NSAIDs may prevent further complications. It is also important to consider any drug-drug interactions before initiating celecoxib or ibuprofen. In general, celecoxib or ibuprofen may be used for pain in opiate addict patients during their detoxifying procedure. Further studies are needed to assess the effects of chronic use of celecoxib and other NSAIDs in opiate addict patients.The study suggests that both celecoxib and ibuprofen improved pain related to opioid withdrawal in the studied patients. Furthermore, no significant difference was observed between the 2 groups in the assessment of alteration of pain perception. On the other hand, the trial revealed a significant difference between celecoxib and ibuprofen on craving for opiates, suggesting a positive evidence for consumption of celecoxib rather than ibuprofen for opiate craving in clinical settings. More randomized controlled trials with larger sample sizes and longer duration of follow- ups are needed to confirm the findings of this study.


It is well established that screening can prevent cervical cancer, but the magnitude of the impact of regular screening on cervical cancer mortality is unknown.N=11 619). Two cancer-free controls were individually age matched to each case. We used conditional logistic regression to estimate the odds ratio (OR) of developing stage-specific cancer for women regularly screened or irregularly screened compared with women not screened in the preceding 15 years. Mortality was estimated from excess deaths within 5 years of diagnosis using stage-specific 5-year relative survival from England with adjustment for age within stage based on SEER  data.Population-based case–control study using prospectively recorded cervical screening data, England 1988–2013. Case women had cervical cancer diagnosed during April 2007–March 2013 aged 25–79 years : 62–73%) reduction in stage 1A cancer and a 95% (95% CI: 94–97%) reduction in stage 3 or worse cervical cancer: the estimated OR comparing regular (⩽5.5yearly) screening to no  screening are 0.18 (95% CI: 0.16–0.19) for cancer incidence and 0.08 (95% CI: 0.07–0.09) for mortality. It is estimated that in England screening currently prevents 70% (95% CI: 66–73%) of cervical cancer deaths ; however, if everyone attended screening regularly, 83% (95% CI: 82–84%) could be prevented.The association between cervical cancer screening and incidence is stronger in more advanced stage cancers, and screening is more effective at preventing death from cancer than preventing cancer itself. Service evaluation of cancer screening is essential to monitor effectiveness, to identify areas of good practice and areas where improvements can be made . The evaThe National Health Service Cervical Screening Programme in England was established in 1988. In the early 1990s, a cervical cancer audit was initiated to evaluate the effectiveness of this programme in preventing cervical cancer. Whereas much has been published on the impact of the programme on the incidence of cervical cancer  smears taken in the United Kingdom since 1988. Local NHS staff linked the screening data to the cases and controls, and the data were pseudonymised locally before being transferred for cleaning and analysis. Guidelines on the collection of data for this audit and details of the design have been published previously  years when women are first screened . Cases with missing stage were therefore proportionally allocated to stages 1B, 2, or 3+, with the proportion in each of these stages determined by the prevalence of the stage in each age group.As age- and stage-specific mortality data are not available for England, we used stage-specific 5-year relative survival from the former Anglia Cancer Network, 2002–2006 , and modThe numbers of cancers and excess deaths within 5 years of diagnosis were estimated: (i) under current screening; (ii) in the absence of screening; and (iii) assuming everyone was regularly screened. For each FIGO stage, the numbers of cancers that would occur in the absence of screening were estimated using Nij is the number of (stage-specific) cases  in age group i with screening category j (see ij is the OR of developing (stage-specific) cancer for women in age group i in screening category j, compared with a baseline category of ‘never screened'. There are three screening categories in addition to the baseline category ; by definition ORi0=1.where The excess number of deaths within 5 years of diagnosis of a specific stage of cancer in the absence of screening was estimated using ρij is one minus the 5-year relative survival in age group i with screening category j (for cancers of the particular stage). As stage 1A cancers are unlikely to be diagnosed in the absence of screening, all stage 1A cancers in never-screened women are assumed in the main analysis to have stage 1B survival. This assumption is relaxed in a sensitivity analysis.where We converted the effect of screening at different ages into absolute numbers of cervical cancer deaths. We assume that the age intervals of death are shifted by 5 years, for example, 25–34 years at diagnosis corresponds to 25–39 years at death, and 35–49 years corresponds to 40–54 years.For the estimated deaths in the absence of/regular screening within age groups, the relative risks (RRs) were multiplied by the observed deaths. The RRs were estimated from the sum of the estimated deaths in each age group when looking at all age groups combined. The confidence intervals (CIs) for these RR were obtained using the appropriately weighted sum of the variance of the individual age groups. Deaths from cervical cancer at all ages were included in this analysis to explore the effect of the screening programme in the population as a whole rather than the effect of screening in the target age group; however, we assume that screening from age 25 to 64 years will not prevent deaths from cancers diagnosed at age under 25 years or age over 79 years.For each age group and a particular level of screening, the RR for incidence  was indirectly calculated by summing the estimated number of cancers (excess deaths) diagnosed across all stages, and dividing by the estimated number of cancers (deaths) in the absence of screening. For comparison, the directly calculated OR for incidence was calculated using a conditional logistic regression model, including all cancers diagnosed in the relevant age group, regardless of stage. As the indirect estimates are weighted sums of the (age and) stage-specific ORs, CIs were estimated using the delta method (for the variance of the log RR), with an (appropriately) weighted sum of the estimated (age and) stage-specific variances  changing the screening window; (ii) assigning cancers with missing stage using a number of criteria; (iii) applying stage 1A mortality to the 1A cancers in the absence of screening scenario; (iv) including women aged 24.5–25.5 years in the analysis. For the sensitivity analyses, we present the OR comparing ‘never'-screened women to the current level of screening in the population  for comparison with Table 4.Analyses were carried out using Stata 13 .A total of 11 619 cases of cervical cancer diagnosed aged 25.5–79 years were included in this study. For 2008–2012, the number of cancers in women aged 25–79 years in the audit was 89.2% of those reported in the Cancer Registry . FIGO stThe estimated 5-year case fatalities  in England by age and stage are shown in We estimate that there would be 2.53 (95% CI: 2.39–2.68) times as many cancers diagnosed aged 25–79 in the absence of screening, and a third less  if everyone was regularly screened. The largest impact of changing screening practices on cancer incidence rates is for women aged 50–64 years, where the incidence rate would be over four times higher with no screening , and less than half  if everyone was regularly screened. Results from the sensitivity analysis  suggest The methodology used to estimate the association between screening and mortality relies on the ability to estimate the stage-specific associations and to combine these to obtain an overall association. We test the robustness of this approach by comparing the OR for cervical cancer (incidence) obtained directly without reference to stage and the RRs obtained by combining stage-specific estimates. The two methods produced very similar results for both point estimates and CIs . The assIn England, there are an average of 796 deaths a year (2011–2014 average) from cervical cancer in women of all ages . It is eIt is generally accepted that quality-assured cervical screening reduces cervical cancer incidence. We have shown that regular cervical screening in England is associated with lower cervical cancer incidence, and that the strength of association increases with age and advancing FIGO stage. Assuming associations are causal, the mortality rate would be 5.3 times higher (95% CI: 4.4–6.4) in the absence of screening or 65% lower (95% CI: 63–67%) if everyone was regularly screened at ages 50–64 years. In summary, if all women attended screening regularly, we estimate that the crude mortality for women aged 25.5–79 years would be half the current (95% CI: 0.48–0.52), whereas it would be 3.6 (95% CI: 3.3–4.0) times higher in the absence of cervical screening. The effect on mortality is greater than the effect on cancer incidence, as cervical screening downstages cancers  as well as preventing them.et al  will (most likely) increase the number of fatal cancers diagnosed at 25 years, even if it reduces (cumulative) cervical cancer mortality. Under the current screening programme, as the population age a large number of deaths that are not preventable by screening will occur after age 84 years, decreasing the relative benefit of screening overall.vs non-screen detected). If anything, it is likely that survival will be better in screen-detected women; if this corresponds to improved cure rates, it would cause our estimates to be conservative. Our model implicitly assumes that stage-specific survival is not affected by route to diagnosis (screen detected The most direct method of evaluating the effect of screening on mortality requires individual-level data on survival following cervical cancer diagnoses, as well as the screening history for those individuals. We know of three such studies. In the absence of individual-level mortality and screening data, it is possible to use trend data to get a very rough estimate of the impact of screening on mortality by looking at the mortality rate before the introduction of cervical screening, and the current mortality rate, incorporating the average screening coverage over this time period. However, this would not allow for differences in treatment efficacy over this time, the differential impact for women who are regularly screened compared with women who are screened infrequently, nor for changes in the underlying rates of cervical cancer in the absence of screening. In addition, large improvements in quality assurance of the programme over the past 20 years have ensured equal access to expert care that has improved survival for all women with cervical cancer .To conclude, we have shown that screening has an even larger impact on cervical cancer mortality than it has on incidence, and that if everyone attended screening regularly, 83% of cervical cancer deaths could be prevented, compared with 70% with current screening. These results are encouraging and should be used to promote, among women, regular attendance to screening and, among policy makers, the implementation of organised screening programmes in areas not yet covered.


To test the feasibility of running a randomised controlled trial of a preconsultation web-based intervention (Presenting Asking Checking Expressing (PACE-D)) to improve the quality of care and clinical outcomes in patients with diabetes.A feasibility study (with randomisation) conducted at outpatient diabetes clinics at two secondary care hospitals in Devon, UK.People with diabetes (type 1 and type 2) attending secondary care general diabetes outpatient clinics.The PACE-D, a web-based tool adapted for patients with diabetes to use before their consultation to generate an agenda of topics to discuss with their diabetologist.The percentage of eligible patients who were recruited and the percentage of participants for whom routine glycosylated haemoglobin (HbA1c) data (the putative primary outcome) could be extracted from medical notes and who completed secondary outcome assessments via questionnaire at follow-up were reported.In contrast with the planned recruitment of 120 participants, only 71 participants were randomised during the 7-month recruitment period. This comprised 18.7% (95% CI 14.9% to 23.0%) of those who were eligible. Mean (SD) age of the participants was 56.5 (12.4) years and 66.2% had type 1 diabetes. Thirty-eight patients were randomised to the intervention arm and 33 to the control arm. HbA1c data were available for only 73% (95% CI 61% to 83%) of participants at the 6 months follow-up. The questionnaire-based data were collected for 66% (95% CI 54% to 77%) of the participants at 6 months follow-up. Participants reported that the PACE-D tool was easy to use.A randomised controlled trial of the preconsultation web-based intervention as set out in our current protocol is not feasible without significant modification to improve recruitment and follow-up of participants. The study also provides insights into the feasibility and challenges of conducting complex intervention trials in everyday clinical practice.ISRCTN75070242. Patients are involved in the development of the intervention.The trial is conducted at two hospital sites.Outcomes are comprised of self-reported patient data collected via questionnaire and routine glycosylated haemoglobin measurements.The patients, clinicians and healthcare staff are not blinded to intervention status.Diabetes is a common chronic condition affecting about 4 million people in the UK and by 2025 it is estimated that the prevalence will rise to over 5 million.3Although diabetes is largely a self-managed condition, specialist healthcare professionals have a vital role to play by helping patients to achieve good diabetes control and to cope with their illness through expert advice, education and support.presenting detailed information about how they are feeling, asking questions if desired information is not provided, checking understanding of information that is given to them and expressing any concerns about the recommended treatment.12Cegala and colleaguesHere, we report results of a randomised controlled trial that we carried out to assess the feasibility of and obtain the necessary information for planning a future definitive trial of this web-based preconsultation intervention (PACE-D) to improve the care quality and clinical outcomes of patients with diabetes. Findings from qualitative work carried out alongside the trial (with the aims of exploring patient and public involvement (PPI) in trial processes, patient experience and relevant organisational factors) are reported in separate papers.This feasibility study used a pragmatic parallel group randomised controlled trial design  with the overall aim of establishing the feasibility of a definitive superiority trial of the PACE-D agenda setting tool.13Patients with diabetes were recruited from two sites  in Devon, South West England. Eligible patients met all of the following criteria: aged 18 years or over; with type 1 or type 2 diabetes mellitus; due to attend for a hospital outpatient appointment with a diabetologist and had sufficient written and spoken English to complete the study assessments. Women with gestational diabetes and patients receiving insulin pump therapy were excluded as these patients are seen in specialised clinics.Potentially eligible patients who were due to attend a general diabetes clinic appointment were identified from clinic lists by healthcare assistants who had been specially trained for the study. Patients who were willing to be contacted were sent an information sheet about the trial and were contacted by phone by the healthcare assistant no less than a week later to confirm eligibility and discuss the study. Patients who wanted to take part in the study were then sent a consent form and baseline questionnaire. On receipt of the signed consent form, the patient was randomised.Participants were randomised in a 1:1 ratio to the intervention or control arms using a computer-generated random allocation sequence prepared by the Peninsula Clinical Trials Unit at Plymouth University (PenCTU). An automated web-based system was used to conceal the allocation. Randomisation was stratified by clinic session using randomly permuted block sizes in a non-systematic sequence. Allocation occurred on receipt of the patient's completed consent form. Following allocation, the hospital informed the patient, via a standard letter, of the time of their clinic appointment. Participants in the intervention arm were asked to attend 30 min before the start of their appointment to ensure sufficient time to complete the PACE-D tool. For both trial arms, if the baseline questionnaire had not been returned to the CTU before the clinic appointment, the participant was asked to complete it in clinic, prior to the consultation (and prior to the PACE-D for those in the intervention arm).https://www.diabetes.org.uk/Guide-to-diabetes/?gclid=CITP94CthdACFQEA0wodVjMJBQ) and their experiential knowledge. A working group of eight people with diabetes was convened by the PPI facilitator. In a 3-hour workshop, this group tested and provided feedback on this early iteration of the intervention. The intervention was fine-tuned using an iterative process.The PACE-D intervention is a web-based tool used by the patient before their consultation to generate an agenda of topics to discuss with their diabetologist. It is based on the PACE tool developed by Cegala and colleaguesThe PACE-D consists of several open and closed questions, prompts and a list of potential concerns the patient might have . A healthcare assistant, trained to facilitate the intervention, was on hand to provide as much help to the patients as required, but without influencing their choices, so that they could go through the PACE-D questions and identify the problems and topics that they wished to discuss in their consultation. After completion of the PACE-D (which takes ∼20 min), a printed personalised consultation agenda was generated for the patient to take into the consultation. The following documents are included as online 10.1136/bmjopen-2016-013519.supp1supplementary materialPatients randomised to the control arm received their standard outpatient appointment with a diabetologist.Data were collected at baseline, 3 and 6 months comprising results of routine measurement of glycaemic control extracted from patient records and several patient-reported measures sent to participants with instructions for completion and a prepaid return envelope. We briefly describe the patient-reported measures. Further details are presented in the protocol paper.13glycosylated haemoglobin (HbA1c) retrospectively obtained from participants' medical records. These data were therefore obtained opportunistically from routine measurements the timing of which was unconnected to the aims of the study. To be used in this study at a given wave, the HbA1c level needed to be measured within 4 weeks of the intended assessment date.Glycaemic control, the putative primary outcome for the definitive trial of the PACE-D tool, was quantified using Audit of Diabetes-Dependent Quality of Life-19 (ADDQoL) measures the patient's perception of the impact of diabetes on their quality of life weighted by its importance to the individual.The Diabetes Empowerment Scale-short form (DES) measures diabetes-related psychosocial self-efficacy.The Diabetes Self-care Activity self-report questionnaire is a measure of diabetes self-management which includes five aspects (activities) of the diabetes regimen: The Diabetes Treatment Satisfaction Questionnaire—Status (DTSQ(S)) measure and change (DTSQ(C)) versions were developed to measure patient satisfaction with diabetes treatment.The Patient Enablement Instrument (PEI) measures patient enablement after a consultation with a physician.et al,The Patient Report of Communication. Developed to measure communication in conjunction with the PACE tool; this instrument comprises 11 questions about perceived communication, with two items for each of the four PACE skills, two additional items for the patient's ability to state their preferences and a global item about the consultation.EuroQoL (EQ-5D-5L) was used to measure generic health-related quality of life. It defines health in terms of: mobility, self-care, usual activities, pain/discomfort and anxiety/depression and uses a 3-point Likert scale .24The Client Services Receipt Inventory was used to collect cost-related and service use-related information for trial participants.The Other outcomes. Participants were asked to document their current medication at each time point, as well as the number and type of contacts with primary and secondary care practitioners during the study. Finally, those in the intervention arm were also asked at 6 months about their experience of using the PACE-D tool and the utility of producing their agenda.It was not possible to blind the participating patients, the consulting diabetologists or the healthcare assistants due to the nature of the intervention which results in the generation of a consultation agenda.The study aimed to recruit 120 patients in total. This is large enough to estimate the percentage that is lost to follow-up at 3 and 6 months with a margin of error no greater than ±9.3% based on the 95% CI width. Assuming that at least 60 patients provide follow-up data, this is large enough to estimate the SD of the continuous outcome measures to within 22% of their true value based on the upper limit of the 95% CI.The main objectives of this trial were to affirm aspects of the feasibility of a definitive trial and estimate parameters for planning recruitment and calculating the sample size for such a study. The percentage of eligible patients who were recruited, the percentage of participants for whom HbA1c data could be extracted from medical notes and the percentage of participants that completed outcome assessments at follow-up are reported with 95% CIs.Characteristics of study participants  and eligible non-participants are summarised using means and SDs for continuous variables and numbers and percentages for categorical variables. The SDs of the outcome measures  are reported with 95% CIs for each trial arm at each of the 3 and 6 month follow-ups.In ancillary analyses, we used the intention-to-treat principle to compare trial arms with respect to the study outcomes  using the t-test for crude estimates of the mean difference and linear regression for estimates adjusted for study site, time since diagnosis and the baseline score for the measure. Ninety-five per cent CIs for the adjusted mean difference are reported, but no p values as this is a feasibility study.The participant flow through the trial is summarised in Three hundred and ninety-five patients with diabetes on the waiting list for appointment in the general diabetes outpatients clinics were screened for eligibility by reviewing their case records; 380 of these were eligible for the trial of whom 71 were recruited, giving a participation percentage of 18.0% (95% CI 14.3% to 22.1%) out of those who were screened and 18.7% (95% CI 14.9% to 23.0%) out of those who were eligible. The number recruited was 59 patients fewer than targeted. Participation was slightly higher in Exeter than Plymouth (24.8% of those who were eligible vs 15.8%). Participants were similar to eligible non-participants with respect to mean (SD) age at registration (56.5 (12.4) versus 51.3 (16.1)), type 1 (as opposed to type 2) diabetes status (66.2% vs 60.5%) and whether attending for a new appointment (4.2% vs 8.1%).Thirty-eight participants were randomised to the intervention arm and 33 to the control arm; the baseline characteristics are summarised by trial arm status in EuroQoL items.Thirty of the 38 participants  in the intervention arm completed the full PACE-D questionnaire and another patient completed only the first of the six parts of the instrument. The median  time taken to complete the PACE-D was 7.1  minutes. Of the intervention participants who responded to questions on how they experienced the PACE-D, 90% ((19/21); 95% CI 70% to 99%) found it to be useful; 84% ((16/19); 95% CI 60% to 97%) found it convenient and easy to use; 81% ((17/21); 95% CI 58% to 95%) thought it helped them think about issues related to their care; 81% ((17/21); 95% CI 58% to 95%) thought it helped them think about their experience of diabetes; 81% ((17/21); 95% CI 58% to 95%) said that it did not increase any stress associated with their appointment and 45% ((9/20); 95% CI 23% to 68%) said that it did change the way the doctor dealt with them. When comparing the length of the consultation, there was little evidence of a difference between the intervention and control arms (mean (SD): 20.2 (12.1) versus 21.8 (8.4) minutes; mean difference: -1.6; 95% CI −8.3 to 5.1; p=0.64).This feasibility study experienced difficulties in recruiting and following up trial participants. Therefore, despite participant reports that the intervention (PACE-D) was acceptable to a majority of patients and was not associated with an increase in the length of the clinic consultation, it would not be feasible to run a definitive randomised controlled trial using the current protocol without further development.This study provides insights into the challenges of conducting a complex trial in everyday clinical practice as shown by the fact that we were able to recruit less than one in five eligible patients into this study. As healthcare team members are often faced with severe time pressures at their work, a key to successful implementation of a new care delivery intervention is to fit in the intervention with existing workflows and cause minimal disruptions on current workflow practices.The majority of patients who completed the PACE-D found the intervention acceptable, easy to use and helped them think about issues related to their care; nearly half of them reported that it changed the way the doctor dealt with them. We recruited people with known diabetes, many of whom had attended the clinics before and some of whom know the clinic staff quite well. Our findings suggest that even for this group, for whom it is more likely that their major questions had already been addressed, the PACE-D tool is potentially useful. We would speculate that newly diagnosed diabetes patients may benefit to a still greater degree.Our findings are consistent with those of a previous study which showed that an online communication tool based on the PACE system is highly acceptable to patients with cancer.A strength of this study is the participation of PPI representatives  at all stages of the study, including proposing the research question, study design, intervention development, training of healthcare assistants, analysis of qualitative data, steering committee membership and preparation of the protocol paper.The study examined several outcomes. Although the Patient Report of Communication measure is designed to capture the impact of the PACE-D, it is questionable whether the other patient-centred outcomes and glycaemic control do. Lack of a theoretical basis for an intervention effect also applies to other potential clinical outcomes not measured here that are relevant to diabetes control, such as lipid levels and blood pressure. The available literature on the pathway between communication and health outcomesThe study suggests that the modified version of the PACE tool was acceptable to patients and helped them to articulate their agendas before an outpatient clinic appointment. It also suggests that this intervention, despite the initial apprehensions of clinical staff, did not lead to longer consultations. Although the study demonstrated that the randomised controlled trial of a web-based preconsultation intervention tool as set out in our original protocol is not feasible, it provides insights into the role of agenda forms in medical consultations. It also provides insights into the difficulties of conducting health services research in clinical settings and the need to have proportionate recruitment procedures for non-clinical interventions. Finally it, adds to our learning about the involvement of PPI representatives in trials of complex interventions.