Internet gaming disorder (IGD) leading to serious impairments in cognitive, psychological and social functions has gradually been increasing. However, very few studies conducted to date have addressed issues related to the event-related potential (ERP) patterns in IGD. Identifying the neurobiological characteristics of IGD is important to elucidate the pathophysiology of this condition. P300 is a useful ERP component for investigating electrophysiological features of the brain. The aims of the present study were to investigate differences between patients with IGD and healthy controls (HCs), with regard to the P300 component of the ERP during an auditory oddball task, and to examine the relationship of this component to the severity of IGD symptoms in identifying the relevant neurophysiological features of IGD. Twenty-six patients diagnosed with IGD and 23 age-, sex-, education- and intelligence quotient-matched HCs participated in this study. During an auditory oddball task, participants had to respond to the rare, deviant tones presented in a sequence of frequent, standard tones. The IGD group exhibited a significant reduction in response to deviant tones compared with the HC group in the P300 amplitudes at the midline centro-parietal electrode regions. We also found a negative correlation between the severity of IGD and P300 amplitudes. The reduced amplitude of the P300 component in an auditory oddball task may reflect dysfunction in auditory information processing and cognitive capabilities in IGD. These findings suggest that reduced P300 amplitudes may be candidate neurobiological marker for IGD. Additional studies to define the features of IGD, to obtain cross-cultural data on the reliability and validity of specific diagnostic criteria and to elucidate its associated biological features are needed before IGD is included in the next version of the DSM as a formal disorder.5The increasing popularity of the Internet has led to a growing body of research in various fields related to Internet addiction, gaming addiction and pathological Internet use.7 Moreover, recent neuroimaging studies have reported significant changes in brain function and structure associated with IGD.10 According to previous research, patients with IGD have reduced regional homogeneity in the superior temporal gyrus at rest.12 The superior temporal gyrus, which contains the primary auditory cortex, is thought be important for integrating auditory and visual information.15People playing Internet games for a long period of time are repeatedly exposed to visual and auditory events, and this continuous exposure to colorful images and dynamic sounds may cause visual or auditory fatigue and problems in related brain regions.18 P300 component of ERP is a large positive deflection that occurs ~300–500 ms after stimuli onset and has maximal amplitude over in the central and parietal regions of the scalp. It is thought to reflect selective attention, memory or processing of incoming information, and has been reported to be reduced in amplitudes in substance use disorder by the majority of studies.19 Several previous studies have provided evidence regarding substance use disorder in the form of ERP data collected during the performance of auditory oddball tasks. Alcoholics showed a significant reduction in the amplitudes of the P300 component, which is deficient in alcoholism, while performing an auditory oddball task.22 Some findings have showed that reduced P300 amplitudes have been observed in individuals at risk for alcoholism, which suggested a lack of capacity to allocate neural resources for encoding specific events and could be due to impaired cortical functions.24 Studies on smoking dependence have also demonstrated a reduction in P300 amplitudes during an auditory oddball task in smokers compared with controls,26 and Moeller et al.27 reported that lower P300 amplitudes were found in cocaine users than in controls.Event-related potential (ERP) measures have been used extensively to investigate brain functions and the neural mechanisms of attention and cognition due to their sensitive temporal resolution and noninvasiveness.29 For example, Dong et al.30 used a go/no-go task to study response inhibition in people with Internet addiction disorder. In particular, as mentioned above, patients with IGD are repeatedly exposed to various kinds of visual and auditory stimulations such that it is necessary to investigate neural functions associated with information processing in IGD. The present study compared the ERP patterns associated with auditory information processing in patients with IGD with those in healthy controls (HCs) to identify the neurophysiological features that may serve as possible biomarkers of IGD. We hypothesized that the P300 amplitudes of patients with IGD in response to target stimuli would be reduced compared with those of HCs. In addition, we hypothesized that there would be the relationship between P300 amplitudes and the severity of IGD symptoms.To our knowledge, no prior study has tested the pattern of the P300 component in patients with IGD using an auditory oddball task, and only a few studies have used ERP methods to examine the features of IGD.31 was used to assess the severity of the participants' disorder. In this study, a modified IAT made for assessing Internet games was used.32 To clarify the pathological changes associated with IGD, we included only those subjects with IAT scores of at least 70 35 and the Barratt Impulsiveness Scale-11 -matched HCs participated in this study. All patients were seeking treatment at the outpatient clinics of SMG-SNU Boramae Medical Center in Seoul, South Korea, due to excessive participation in Internet gaming. The Institutional Review Board of the SMG-SNU Boramae Medical Center approved the study protocol, and all subjects provided written informed consent before participation. A clinical interview by an experienced psychiatrist was administered for the diagnosis of IGD according to DSM-5 criteria, and Young's Internet Addiction Test (IAT) 70 ref.  who spen-11 ref.  were useExclusion criteria were a history of significant head injury, seizure disorder, mental retardation, psychotic disorder and substance use disorder except nicotine. All participants were medication-naive at the time of assessments. The Korean version of the Wechsler Adult Intelligence Scale-III was administered to all subjects to estimate IQ, and we included only subjects with Wechsler Adult Intelligence Scale-III scores of at least 80.We used the auditory oddball task, which involves presenting standard stimuli (85%) as well as rare, deviant stimuli (15%) in a pseudorandomized order at an 85-dB sound pressure level. Three hundred stimuli were presented binaurally by a STIM 2 sound generator . Stimuli were presented under two different pitch conditions: the infrequent deviant stimulus was classified as a high-frequency tone (2000 Hz); and the frequent standard stimulus was classified as a low-frequency tone (1000 Hz). The duration of each tone was 100 ms  with fixed intertrial intervals of 1250 ms. The participants were instructed to press a response-box button with their right hand as quickly and accurately as possible in reaction to only to high tones. All participants were given the opportunity to practice before the actual task started. Participants completed three blocks of 100 trials while seated in a comfortable chair.Electroencephalogram and electrooculogram data were recorded using a 64-channel Quick-cap system (Compumedics) that referred to the linked mastoid in an isolated sound-shielded room. The location of the ground channel was between FPz and Fz. Horizontal and vertical electrooculograms were measured by electrodes placed at the outer canthus of each eye, and above and underneath the left eye, respectively. The electrical activities were continuously recorded at a sampling rate of 250, 500 or 1000 Hz. A band-pass filter was set to 0.3–100 Hz. Impedance at all recording electrodes was <10 kΩ.et al.37 Data were then segmented into epochs of 1000 ms, which included the 100-ms pre-stimulus baseline period. Epochs with voltage in excess of ±70 μV were discarded automatically. Only trials with correct responses to deviant tones at four midline sites  were averaged and analyzed. Midline electrodes are commonly selected in oddball tasks investigating P300 components. The ERP waveforms for each participant had a minimum of 35 artifact-free trials. The P300 component was defined as the largest positive-going peak within the time window between 248 and 500 ms after stimulus onset. Topographic maps of P300 amplitudes were created using Scan 4.5 software (Compumedics).Electrophysiological signals were further processed off-line using Curry 7 software (Compumedics). Recordings were first downsampled into 250 Hz. Data were then re-referenced against the common average reference and filtered with a frequency band pass from 0.3 to 30 Hz. Electroencephalogram and electrooculogram recordings were visually inspected to reject gross artifacts such as those involving movement. Eye blinks and eye movements were corrected based on the method of artifact reduction developed by Semlitsch χ2-test, with the treating group (IGD and HC) as the between-subject factor. In terms of stimulus-locked ERP values, the amplitudes and latencies of the P300 component were separately analyzed with repeated-measures ANOVAs with electrode sites  as within-subject factors and group as the between-subject factor. In the case of sphericity violations, the lower-bound corrections were applied, and the corrected P-values were reported. P300 values associated with significant intergroup differences were subjected to analyses with clinical variables using two-tailed Pearson's correlation coefficients. Results with P-values <0.05 were regarded as significant. The variables showing significant main effects were further analyzed with post hoc comparisons using one-way ANOVAs. All statistical analyses were performed using SPSS v18.0 software .Demographic, clinical and behavioral data were analyzed with one-way analyses of variance (ANOVAs) or =450.99, P<0.001), BDI =49.92, P<0.001), BAI =11.17, P<0.01) and Barratt Impulsiveness Scale-11 =57.50, P<0.001) compared with HCs. The demographic and clinical characteristics of participants are presented in No significant group differences existed with regard to age, sex, education and estimated IQ. Patients with IGD had significantly higher scores on the IAT =16.73, P<0.001) and group =4.69, P=0.029) for P300 amplitudes were found. The P300 amplitude measured at CPz was the highest among the four electrode sites. No significant interaction was observed between the electrode site and group for the P300 amplitudes. Patients with IGD showed significantly lower P300 amplitudes than HCs at CPz =8.02, P<0.01) but not at FCz, Cz and Pz. In terms of P300 latencies, none of the main effects or interactions was statistically significant.The grand-average ERP waveforms for deviant stimuli at four electrode sites are shown in r=−0.324, P=0.025). No significant correlations were found between P300 amplitudes and BDI, BAI and Barratt Impulsiveness Scale-11 scores.Significant correlations were found between P300 amplitudes and IAT scores . IAT sco39We investigated the electrical brain activity using an auditory oddball task in response to deviant stimuli. The auditory oddball task in this study may have been too easy, and behavioral performance did not differ significantly between patients with IGD and HCs. However, the present study found ERP differences between the two groups in an auditory oddball task. Therefore, ERP differences between groups were not due to differences in the behavioral performance but neurophysiological changes in the IGD group. Consistent with our prediction, the amplitude of the P300 component in response to deviant tones was diminished in patients with IGD compared with HCs at the midline centro-parietal electrode region. These reductions in the P300 amplitude in the auditory oddball task indicate that patients with IGD suffer from a dysfunction in auditory information processing and cognitive functioning. These results are consistent with previous ERP studies with individuals suffering from other addictions, who exhibited a decrement in the P300 amplitude.40 Therefore, the P300 component indexes fundamental attention- and memory-related operations. The correlational analysis revealed significant relationships between reduced P300 amplitudes and severe IGD symptoms. These results suggest that the P300 amplitude changes observed could be related to the clinical status of IGD and may be a candidate neurophysiological marker of IGD.P300 is considered to reflect the information-processing cascade associated with attentional and memory mechanisms. If the incoming stimulus in the oddball task is not the same and the subject allocates attentional resources to the target, the neural representation of the stimulus environment is updated and a P300 is elicited in addition to sensory potentials.42 Although the precise neural origins of P300 are not clearly understood, some studies have consistently found that the P300 component is produced by a neural circuit pathway between the frontal and temporo-parietal regions.44 The classic P300 component typically refers to the P3b elicited by target stimuli, whereas another subcomponent of the P300 is the P3a elicited by novel or nontarget stimuli. The P300 used in the present study refers to the P3b. The P300 component (or P3b) may originate from temporo-parietal regions, and the P3a may originate from frontal regions.46 Kim et al.12 reported resting-state functional brain changes in the superior temporal gyrus and the posterior cingulate cortex in patients with IGD. As the posterior cingulate cortex is part of the default mode network as well as the parietal region, which is synchronized with low-frequency oscillations in some brain regions during the resting state, it is important for attention and self-monitoring as it involves cognitive functions associated with the coupling of executive control and the ability to disengage from the default mode network.47 The superior temporal gyrus is thought to be important in the processing of audiovisual information and is also among the key regions involved in the integration of auditory and visual cues, and in emotional perception based on auditory/visual information.15 The reduced P300 amplitudes in patients with IGD found in the present study may represent neurophysiological changes in the temporo-parietal regions, which are consistent with previous findings.12 In addition, this finding indicates that changes in P300 amplitudes may be associated with repeated exposure to various kinds of visual and auditory stimulations during playing Internet games, in patients with IGD.The neural generators of the P300 component have been widely investigated.49 On the other hand, Polich and Criado50 reported that the P300 amplitudes of controls and patients with restless leg syndrome were comparable, but they were greatly diminished in patients with Parkinson's disease, who have low levels of dopamine in the brain. Pogarell et al.51 also found that striatal dopamine D2/D3 receptor status was positively correlated with the P300 amplitudes in response to target tones in patients with depression. That is, reduced P300 amplitude is associated with reduced dopaminergic activity. Several previous studies have suggested that Internet addiction or IGD is associated with abnormalities in the dopamine reward system. Kim et al.52 found reduced levels of dopamine D2 receptor availability in subdivisions of the striatum, including the bilateral dorsal caudate and right putamen, in individuals with IGD. Abnormalities in the P300 amplitudes of patients with IGD may be indicative of impaired dopaminergic systems in IGD, which are commonly observed in other addiction disorders.53Although the exact neurotransmitter systems underlying P300 generation remain unclear, some lines of evidence imply a neurotransmitter mediation of P300 generation. With regard to the P3b component, norepinephrine activity, which originates in the locus coeruleus, may contribute to P300 (or P3b) generation in humans.55 Therefore, our results excluded the effect of medication on ERPs. Second, patients with IGD had significantly higher scores on the BDI and BAI compared with HCs. To control potential confounding effects, analyses of covariance with BDI and BAI scores as covariates were performed on P300 amplitudes, and significant differences in P300 amplitudes still maintained between two groups. In addition, when we did analyses in individuals with IGD after excluding those with depressive disorder or anxiety disorder, the results were still significant. Moreover, we found no significant correlation between the P300 amplitude and the BDI and BAI scores. Third, the IAT scale used for the assessment of severity of IGD was a self-report form, which could lack objective characteristics. Fourth, a cross-sectional design was used in this study, but a longitudinal study observing the same participants over time would be more beneficial for elucidating the development of this disorder. Finally, we did not have clinical control group such as substance use disorder. In the further study, it is necessary to compare those in IGD with other addictive disorders in order to clarify the neurophysiological features specific to IGD. Despite these limitations, the findings of this study contribute to our understanding of alterations in the P300 component and the association of this component with the neuropsychological deficits associated with IGD.The present study has several limitations. First, the sample used in this study was small, limiting the generalizability of the findings. Thus, future studies with larger samples are needed to more confidently identify the features of IGD. Although the number of participants in the study was small, we controlled for demographic characteristics such as age, sex, education, IQ and medication status. None of the participants was receiving medications. Electrophysiological activity can be affected by medications.In conclusion, our results reflected a reduction in the P300 amplitudes of the IGD group compared with those of the HC group during an auditory oddball task. Furthermore, the reduced amplitude of the P300 component was negatively correlated with the severity of IGD, which may represent deficits in auditory information processing and cognitive functions, as well as the relationship between this component and pathological Internet game use. The findings of the present study suggest that the reduction in P300 amplitude associated with brain functional abnormalities may be a candidate neurobiological marker for IGD, which may provide further insight into the neurophysiological mechanisms underlying this disorder. In order to identify whether alterations of P300 amplitudes in patients with IGD could be considered as a candidate trait maker or state maker, additional longitudinal studies and analysis of P300 amplitude in subjects at high risk for IGD are expected. When the abnormalities of P300 could be present in populations at high risk for IGD, P300 component of ERP could be regarded as a trait maker for IGD. In addition, when the abnormalities of P300 could be normalized along with symptom improvements after longitudinal assessments in patients with IGD, P300 indexes could be regarded as a state marker for IGD. Then, it could be used for assessing prognosis of IGD, or for prevention and early therapeutic interventions in patients with IGD.


To determine (1) reconsultation frequency, (2) change in self-reported health status, (3) baseline factors associated with reconsultation and change in health status and (4) associated healthcare costs and quality-adjusted life-years (QALYs), following assessment at a musculoskeletal Clinical and Assessment Treatment Service (CATS).Prospective cohort study.Single musculoskeletal CATS at the primary–secondary care interface.2166 CATS attenders followed-up by postal questionnaires at 6 and 12 months and review of medical records.Primary outcome was consultation in primary care with the same musculoskeletal problem within 12 months. Secondary outcome measures were consultation at the CATS with the same musculoskeletal problem within 12 months, physical function and pain (Short Form-36), anxiety and depression , time off work, healthcare costs and QALYs.Over 12 months, 507 (38%) reconsulted for the same problem in primary care and 345 (26%) at the CATS. Primary care reconsultation in the first 3 months was associated with baseline pain interference  and spinal pain , and after 3–6 months with baseline assessment by a hospital specialist . Small mean improvements were seen in physical function  and body pain  at 6 months. Poor physical function at 6 months was associated with obesity, chronic pain and poor baseline physical function. Mean (SD) 6-month cost and QALYs per patient were £422.40 (660.11) and 0.257 (0.144), respectively.While most patients are appropriate for a ‘one-stop shop’ model, those with troublesome, disabling pain and spinal pain commonly reconsult and have ongoing problems. Services should be configured to identify and address such clinical complexity. The largest study to date of outcome following treatment in a musculoskeletal Clinical and Assessment Treatment Service.The participation rate at baseline was high and the use of routinely collected consultation data ensured high completion rates for the primary outcome.Response to the postal follow-up questionnaires was poor, particularly at 12 months.Questionnaire length permitted inclusion of only generic measures of pain and physical function rather than body region-specific measures which might have been more sensitive to improvement.The study population was derived from a single geographical region and did not include a comparator cohort which might limit the generalisability of our findings.Musculoskeletal problems such as osteoarthritis (OA) and back pain are highly prevalent and present frequently to primary care. One-third of adults experience low back pain annually, whereas 53% of older adults have symptomatic OA.4Most of these people are managed entirely in primary care, with only a minority requiring specialist referral, traditionally to hospital-based orthopaedic and rheumatology services. Recently, patients requiring referral in the UK are increasingly managed in multidisciplinary Clinical Assessment and Treatment Services (CATS) at the primary–secondary care interface.The objectives of this prospective study were (1) to determine the proportion of patients reconsulting in primary care and in a musculoskeletal CATS in the 12 months following baseline assessment at the CATS, (2) to assess baseline factors associated with reconsultation in primary care and at the CATS, (3) to determine change in self-reported health status at 6 and 12 months, (4) to assess baseline factors associated with change in self-reported health status and (5) to estimate the healthcare costs and quality-adjusted life-years (QALYs) over 6 months associated with CATS attendance and determine whether these costs and QALYs differed by follow-up plan at baseline.This was a prospective observational study set within a musculoskeletal CATS in North Staffordshire, UK. The methods and baseline cross-sectional findings have been described previously.9At the time of baseline data collection, Stoke-on-Trent Primary Care Trust (PCT) served a population of >270 000 people. Referrals to secondary care musculoskeletal, rheumatology and orthopaedic services are triaged by clinicians to a multidisciplinary, musculoskeletal CATS at the primary–secondary care interface following review of referral letters, so that musculoskeletal conditions requiring non-surgical interventions are managed in the community, while appropriate cases are directed to rheumatology or orthopaedic services. The CATS is the preferred provider for patients with non-surgical, non-inflammatory musculoskeletal problems. Patients are triaged to unselected general musculoskeletal clinics within the CATS, where the type of healthcare professional patient seen ) and the clinic in which they are seen are not determined by the index referred condition. The sole exception to this is a physiotherapist-led back pain clinic. A greater proportion of patients with back pain therefore see a physiotherapist compared to other conditions.All adults aged ≥18 years seen at the CATS between February 2008 and June 2009 were invited to participate. Those who consented to take part completed a health questionnaire prior to their CATS appointment. Participants were also asked to provide consent for the research team to review their medical records, which provided data to answer the primary outcome of consultation in primary care with the same musculoskeletal problem within 12 months.The questionnaire included physical functioning and body pain scales from the Short Form-36 (SF-36) V.2 .15The clinician conducting the CATS consultation completed a brief proforma but did not see the patient's completed questionnaire. The clinician proforma recorded the location of pain addressed in the consultation , investigations, interventions, referrals and follow-up plan. Participants were regarded as having pain in multiple sites if the clinician recorded locations from two or more of the upper limb/neck, spine or lower limb, or recorded a diagnosis of fibromyalgia, chronic widespread pain, generalised OA or polymyalgia rheumatica. Follow-up was categorised as referral to other services , CATS follow-up, discharge to the GP or to be decided following investigations. If the follow-up plan was dependent on investigation results, follow-up information for those participants who consented to medical record review was extracted from CATS records. The clinician profession was also recorded. Owing to the low number of rehabilitation medicine specialists, we combined these with rheumatologists . We also recorded whether patients were attending a general musculoskeletal clinic or the physiotherapist-led back pain clinic described above.A self-administered questionnaire containing the same measures as at baseline was mailed at 6 and 12 months to all consenting participants. Non-responders were sent a postcard reminder after 2 weeks and a repeat questionnaire after 4 weeks.In participants consenting to medical record review, information was extracted from primary care records for the 12 months following baseline. Owing to the large number of general practices (n=114 including 49 with fewer than 10 patients), it was unfeasible to examine the records of all patients and so a pragmatic decision was made to extract records from the 57 most accessible practices with the highest number of participants. Records were downloaded electronically where possible but where software was incompatible with the practice, information was extracted manually using a proforma. The information extracted was date of any musculoskeletal consultation in primary care, and date of a musculoskeletal consultation for the same body location  as recorded by the clinician at baseline. Musculoskeletal consultations were identified using the Read code system which is commonly used to record morbidity in UK primary care.At the time of baseline data collection, ∼3500 patients were seen in the CATS annually. Based on previous studies, we expected 75% of these to participate at baseline (n=2625), and 75% of these to separately consent to further postal contact and medical record review (n=2000 each). While we aimed to review records of as many patients as feasible, as an example, a sample size of 1125 is sufficient to determine the percentage making a repeat primary care consultation during 12 months follow-up with a margin of error of 3% and a 95% confidence level, based on an estimate of 50%.We compared baseline responders with extracted medical record data with all other baseline responders on baseline sociodemographic, pain characteristics and general health. The percentage consulting for a musculoskeletal problem in primary care in the 12 months after baseline was determined. The primary analysis was based on the time to consulting in primary care about the same body location which was addressed at the baseline CATS consultation. We split time to first consultation to primary care into no consultation during 12-month follow-up, first consultation within 3 months (early), between 3 and 6 months, and between 6 and 12 months after baseline clinic assessment (late). We used these categories rather than actual time as it was evident that the baseline factors associated with first primary care attendance changed over the 12-month time-period, and it was considered that attendance to primary care within 3 months may be at the request of the CATS clinician. Multinomial logistic regression was used to determine the association of follow-up plan and clinician profession with the time of consultation to primary care adjusted for self-reported  and clinician-reported  factors. No repeat consultation was treated as the reference category. Results are reported as adjusted relative risk ratios (RRR) with 95% CIs.Secondary outcomes were reconsultation at the CATS during 12-month follow-up and self-reported health  at 6 and 12 months. Binary logistic regression was used to assess association of clinician profession and follow-up plan with reconsultation at the CATS about the same body location as at the baseline clinic at any point during the 12 months, adjusting for the same factors included in the primary outcome analysis. Results are presented as ORs with 95% CI. Multiple linear regression was used to assess the association of clinician profession and follow-up plan with physical function score at follow-up adjusted for baseline score and for the same baseline self-reported and clinician-reported factors as included in the analysis of primary care consultation .Two sensitivity analyses were performed. First, as primary care medical record information was not available for everyone, we performed multiple imputation with 50 imputations and again repeated the analysis. Second, because of the attrition at follow-up, the analysis of self-reported physical function at 6 months was repeated using multiple imputed data for those not responding at follow-up.The health economic analysis was conducted from a healthcare perspective and focused on estimating the costs and QALYs arising from attending the CATS. Resource use data were collected from the clinician proforma and 6-month questionnaire. The proforma recorded investigations and interventions that patients received, while the questionnaire asked about the number and type of health professionals seen, medication taken and the number of interventions. Unit costs for individual resource use items were obtained from sources such as the British National Formulary (BNF), Personal Social Services Research Unit and NHS reference costs.As reported previously,10.1136/bmjopen-2016-011735.supp1supplementary figureFlow of participants through the study10.1136/bmjopen-2016-011735.supp2supplementary tablecomparison of baseline characteristics between those included and excluded from primary care record analysisOf the 1453 for whom record data were collected, 1342 were included in the primary outcome analysis as the remainder received other diagnoses such as gout, inflammatory arthritis and joint hypermobility, and hence, a specific body region was not available to link subsequent consultations to  reconsulted at the CATS during 12-month follow-up for a musculoskeletal problem in the same body location as assessed at baseline. The clinician stating they would follow-up the patient  was most strongly associated with reconsultation at the CATS while being discharged  was also significantly associated . PatientOne thousand one hundred and forty-three (54%) of the 2116 baseline responders with a completed clinician proforma completed the follow-up questionnaire at 6 months and 762 (36%) at 12 months. Six-month responders were older (mean age 54.9 vs 46.4) compared to non-responders and had lower levels of anxiety and depression. However, they did not differ according to pain, physical function or the type of clinician seen at baseline. Responders showed some improvement at 6 months in body pain , whereas a smaller change was seen in physical functioning  . The perType of clinician seen and follow-up plan did not associate with physical function at 6 months . FemalesThe overall mean (SD) cost per patient incurred by attending the CATS was £422.40 (660.11) over the 6-month period. The mean cost associated with patients who were discharged was significantly lower than those who were referred to other specialities  . Costs aAcross all participants, the mean (SD) QALYs per patient over the 6-month period was 0.257 (0.144). There was no significant difference in mean QALYs between patients who were referred to other specialities and any other group.After assessment in a musculoskeletal CATS, nearly 40% of people consulted primary care about the same problem within 12 months, with over half of these consulting within 3 months. Similarly, a quarter of patients reconsult in the CATS within 12 months. People with pain interference and spinal pain were more likely to reconsult. Over 6 months, only small changes were seen in body pain and physical function, and in the proportion reporting pain interference and taking time off work because of their musculoskeletal problem. Functional outcome was worst in those with older age, obesity, chronicity and pre-existing physical impairment. The cost-outcome description found that follow-up plan to see again in the CATS or to refer to another specialty attracted higher mean costs.The explanations for frequent reconsultation are likely to be multifactorial, and our study design cannot elucidate these. It may be that patients were advised to visit their GP for change in medication, they reconsulted to obtain a repeat prescription, the CATS consultation failed to adequately meet patients’ expectations and/or their symptoms did not improve. The observation that patients assessed by hospital specialists were more likely to reconsult could be explained by specialists advising further consultation in primary care, for example, to change medication, rather than this reflecting poor outcome. The findings that people with pain interference (either setting) and spinal pain (primary care) were more likely to consult and worse functional outcome was associated with older age, obesity, chronicity and pre-existing physical impairment suggest that the current model of care does not meet the needs of those with the most troublesome symptoms. An unexpected finding that we find difficult to explain was that people with anxiety or depression were less likely to first consult primary care between 6 and 12 months.There are few suitable cohorts to compare our findings to. In a study undertaken in a physiotherapist-led CATS, small improvements were reported in pain and general health (EQ-5D) over 12 months but no change was seen in the SF-36.24This is the largest study to date of outcome following treatment in musculoskeletal CATS. Strengths of the study are the high participation rate at baseline (73%) and the use of routinely collected consultation data to ensure high completion rates for the primary outcome. Several limitations are, however, worthy of further discussion. First, the response to the postal follow-up questionnaires was poor, particularly at 12 months. However, questionnaire data were used to answer the secondary objectives rather than the primary objective which used data from consultation records, available for 69% of baseline responders. Furthermore, a sensitivity analysis using multiple imputation to account for loss to follow-up did not significantly alter our findings. Second, pain and physical function were measured using generic health status instruments, finding only small changes over time. Owing to questionnaire length, we could not include body region-specific questionnaires which might have been more sensitive to improvement. Third, the study population was derived from a single geographical region and participants recruited from a single CATS which might limit the generalisability of our findings. Finally, we did not include a comparator cohort to allow a direct comparison to patients managed in other settings such as primary care, orthopaedics or rheumatology.Our findings suggest that musculoskeletal CATS should be configured to address troublesome disabling pain since it is patients with the most bothersome symptoms who are most likely to reconsult either in primary care or at the CATS and to experience poor functional outcome. We have previously highlighted the complexity of patients referred from primary care to musculoskeletal CATS, showing chronic pain, major physical limitation, anxiety, depression and work disability to be highly prevalent.


Studies linking mutations in Methyl CpG Binding Protein 2 (MeCP2) to physiological defects in the neurological disease, Rett syndrome, have focused largely upon neuronal dysfunction despite MeCP2 ubiquitous expression. Here we explore roles for astrocytes in neuronal network function using cortical slice recordings. We find that astrocyte stimulation in wild-type mice increases excitatory synaptic activity that is absent in male mice lacking MeCP2 globally. To determine the cellular basis of the defect, we exploit a female mouse model for Rett syndrome that expresses wild-type MeCP2-GFP in a mosaic distribution throughout the brain, allowing us to test all combinations of wild-type and mutant cells. We find that the defect is dependent upon MeCP2 expression status in the astrocytes and not in the neurons. Our findings highlight a new role for astrocytes in regulation of excitatory synaptic signaling and in the neurological defects associated with Rett syndrome. New roles played by astrocytes in nervous system functioning continue to emerge. However, many questions related to the mechanisms underlying astrocyte-neuronal communication, as well as to the general importance of this communication, still remain unanswered. Patch clamp electrophysiology, especially the ability to make simultaneous measurements from the astrocyte and the neuron, provides a sensitive means to probe their interactions. Here, we use dual patch clamp recordings to extend our understanding of astrocyte-mediated modulation of synaptic transmission within the cortical network. This approach also provides a unique opportunity to determine the potential consequences of defective astrocyte-neuronal signaling in a neurological disease, using a mouse model for Rett syndrome (RTT).Mecp2 from the neuronal tau locus in mice prevented the appearance of some RTT-like phenotypes  wild-type mice . An astrWhen held at −80 mV, both neurons and astrocytes could be further distinguished by spontaneous synaptic events . NeuronaDepolarization of the astrocytes using 20 consecutive 10 ms voltage steps from −80 mV to +60 mV led to an increase in the frequency of the synaptic currents in the proximal, paired neuron . The inchttp://astrocyternaseq.org/addgene?query=F2r), and protein levels . Par1 is expressed in astrocytes at both the mRNA (n levels . In our n levels . Par1 isn levels .50 = 1.9 μM) to wild-type astrocytes consistently resulted in an increase in synaptic event frequency in the proximal neuron recording, qualitatively similar to that seen with astrocyte depolarization , rendering both excitatory. To test whether the GABAA receptors (GABAAR) are excitatory or inhibitory under native transmembrane chloride gradients, we recorded GABA-activated responses using the perforated patch technique with the chloride impermeant gramicidin as the pore forming antibiotic , thereby creating inwardly-directed glutamate and outwardly-directed GABA activated currents  . GABA-meAR activation for initiation of glutamatergic transmission. By contrast, the application of TFLLR in the presence of the glutamate receptor blockers, AP5/NBQX, failed to invoke statistically significant increases in either inward currents  or outward currents   , but lef n = 15) . This reAs a further test of direct release of glutamate or another ionotropic modulator from astrocytes, we tested for any synaptic currents in the proximal neuron that were synchronized to astrocyte stimulation . The expHaving established the characteristics of astrocyte-mediated activation of neuronal signaling in wild-type mice, we performed paired patch clamp recordings on cortical slices from male MeCP2 globally null mice. The effects of depolarization were examined first . RepreseNext, TFLLR was tested for its ability to activate neuronal signaling in slices from MeCP2 null male mice . RepreseTo examine more closely for changes in either GABA- or glutamate-mediated event frequency in MeCP2 null recordings, the frequencies were again parsed into inward and outward currents by use of lowered internal chloride. As found with symmetrical chloride, recordings from MeCP2-deficient slices showed no significant increases in either inward or outward current frequency in response to TFFLR : outward: pre-TFLLR: 25 ± 11 events/m, post-TFLLR: 20 ± 9 events/m, p=0.09, n = 6) .Mecp2 gene. In this line, MeCP2-GFP is expressed in ~50% of all neurons and astrocytes, whereas the remaining 50% of the cells express a truncated, nonfunctional MeCP2 protein  or a calcium-BAPTA buffer corresponding to 100 nM free calcium (n = 4). After 15 min of BAPTA dialysis via the astrocyte patch pipette, TFLLR (500 μM) was applied locally while the frequency of events was determined for a proximal neuron. Dialysis with either BAPTA-based buffer reduced the basal frequency of neuronal events from the collective pre-stimulus levels by 0.72 ± 0.08 fold, along with no further increase in frequency following TFFLR application  . This deD = 9.7 μM) for 35 m, which preferentially loaded SR101 positive astrocytes. In all 23 wild-type astrocytes tested, somatic increases in calcium were observed in response to TFLLR application  (p=0.012)  .2) for 10 m via the patch pipette. Calcium was then uncaged in the astrocyte via a 1 s LED light flash while the frequency of synaptic currents in the neighboring neuron was recorded  and MeCP2 null animals   In wild-type slices, uncaging of calcium loaded DMNP-EDTA resulted in an average 1.39 ± 0.08 fold increase in the frequency of synaptic currents in the neighboring neuron  . In contThere is precedent from both electrophysiological recordings and calcium imaging for the ability of astrocyte stimulation to affect neuronal function in the brains of healthy mice . The mos3-mediated release of calcium through the endoplasmic reticulum. The Par1 receptor expresses at high levels in astrocytes  of the metabotropic Protease-activated receptor 1 (Par1). Activation of the astrocyte via Par1 works primarily through Gα signaling, leading to IPtrocytes , a finditrocytes . Evidenctrocytes . Thus, wAR involvement in the initiation of the circuitry. By contrast, applying glutamate receptor antagonists blocked the TFLLR induced increase in the majority of outward GABA mediated responses and most, but not all of the glutamate mediated inward responses. The persistence of inward responses could reflect an incomplete block of glutamate receptors or involvement of additional unidentified chemical activators. As a further test for involvement of a direct glutamate receptor response we performed time-locked neuronal recordings in response to astrocytic depolarization. Examination of the pooled event timing revealed a significant, but delayed, increase in frequency within 40 to 60 ms of astrocyte depolarization. These responses were further tested for evidence of direct activation through inclusion of TTX which blocks neuronal mediated effects. TTX eliminated the increase in event frequency associated with the depolarization pointing to release from neurons and not astrocytes. Therefore, the putative molecule/s underlying astrocyte modulation of neuronal signaling in our context is still at large.A central question that surrounds astrocyte effects on neuronal excitability is the identity of the factor/s released causal to the initiation of signaling. The list of published candidates includes glutamate , ATP Ne, D-serin2 chemosensitivity  synaptic balance reported in RTT . Normallelopment . This swelopment . In bothelopment . Consequtm1.1Bird , Mecp2 mice were crossed to heterozygous Bnull/+Mecp2 mice.All animal procedures were approved by the Oregon Health and Science University Institutional Animal Care and Use Committee under protocol number IP00000284. Mice were kept on 12 hr light/dark cycles and were housed with their littermates. Three genotypes were used in this study: C57BL/6J,  003890; ), and Me 014610; ). All mi2PO4, 1.3 Na-ascorbate, 3 Na-pyruvate, 10 glucose, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2 and adjusted to pH 7.3. Slices were then placed in a recovery solution for 35 m at 35°C which contained: (in mM) 119 NaCl, 2.5 KCl, 1 NaH2PO4, 1.3 Na-ascorbate, 3 Na-pyruvate, 10 glucose, 26 NaHCO3, 1.5 CaCl2, 1 MgCl2 and 100 nM SR101 to label astrocytes. The recovery solution was continuously bubbled with Carbogen . Slices were then transferred to a lower temperature recovery solution (23˚C) for an additional 35 m and used within 5 hr.Mice between ages p10 and p12 were anesthetized with isoflurane prior to decapitation and removal of the brain. Coronal slices (200 μm) were cut from the barrel cortex of the right hemisphere using a vibratome (Leica VT1200s). An ice-cold cutting solution contained: (in mM) 110 CholineCl, 2.5 KCl, 1.25 NaHAll recordings were performed at ~32°C from layer 2/3 barrel cortex astrocytes and neurons. Astrocytes and neurons were visualized by means of an IR-DIC 40x W achroplan objective using a Zeiss Axiocam MRm . Astrocytes were identified on the basis of Sulforhodamine 101 red fluorescence . In MeCP2-GFP knock-in mice, expression of MeCP2 was identified on the basis of green fluorescence.2PO4, 10 glucose, 26 NaHCO3, 2 CaCl2, and 1.3 MgCl2, The recording solution was continuously bubbled with Carbogen to maintain the pH at 7.3. The internal solution for symmetrical chloride recordings contained: (in mM) 140 CsCl, 10 Cs-HEPES, 10 Cs-EGTA, pH 7.35. The internal solution for low intracellular chloride contained: (in mM) 130 K-Gluconate, 10 KCl, 10 K-EGTA, 10 K-HEPES, pH 7.38. For test for a calcium requirement in signaling the patch pipette contained either 10 mM BAPTA  or 10 mM BAPTA with 4.83 mM CaCl2 . All recordings utilized a dual headstage EPC10 HEKA amplifier. Data was acquired using Patchmaster software  at a sampling rate of 10 kHz and Bessel filtered offline at 500 Hz.The extracellular recording solution contained: (in mM) 110 NaCl, 2.5 KCl, 1 NaHFor perforated patch neuron recordings, the tip of the patch pipette was filled with gramicidin-free internal solution prior to backfilling with internal solution containing 100 μg/mL gramicidin . Following seal formation the resistance was monitored until it dropped to <50 MΩ. Establishment of the whole cell recording was then confirmed on the basis of a large voltage activated sodium current.The amplitude and frequency of individual synaptic currents were determined using a combination of Patchmaster, ClampFit  and Mini-analysis software . After 500 Hz filtering the detection threshold was set to 3x RMS noise for symmetrical chloride recordings and to 2x RMS for low chloride internal solution recordings. Statistical analyses were carried out using GraphPad Prism6 software . The paired t-tests used either a parametric paired-t test or the Wilcoxon matched-pairs signed rank test and the unpaired t-test used the Mann-Whitney test. All t-tests were two-tailed and a confidence level of 95% was used. Statistical significance was based on p<0.05.2, and GABA were pressure applied through a patch electrode via a picospritzer . The antagonists gabazine (SR95531 hydrobromide), DL-AP5, and NBQX were incubated with the slice for minimum of 10 m included in puffer pipette along with the appropriate agonist. All agonists and antagonists were obtained from Tocris . Tetrodotoxin (TTX) was obtained from Alomone Labs The agonists L-glutamic acid, TFLLR-NH0 as follows. First, the average pixel intensity within each ROI was corrected for background fluorescence. To establish true background levels the average pixel intensity was determined using cells not incubated with the calcium indicator. Second, the background corrected basal fluorescence signal intensity (F0) was determined on the basis of the average pixel intensity measured for the ROI during the two seconds prior to drug application. Third, the change in average fluorescence intensity for the ROI (ΔF) was determined by subtracting the average pixel intensity for the ROI by the background-subtracted basal fluorescence (F0).Astrocytic somatic calcium signals were quantified using 10 μM Fluo4FF-AM . After recovery at 35˚C, slices were incubated in extracellular recording solution containing equal volumes of calcium indicator and 20% (w/v) Pluronic acid . These slices were maintained at 23˚C for 25 m during which they were shielded from light and continuously bubbled with Carbogen. Slices were then transferred to the microscope stage and washed for 30 additional minutes with bubbled extracellular recording solution to allow for de-esterification of the calcium indicator. Live calcium signals were acquired using a Yokogawa CSU-X1 confocal spinning disc equipped with a Hamamatsu EM-CCD digital camera (Imagem-x2). A 488 nm laser line was used for calcium signals and 561 nm line was used for SR101 fluorescence . Laser power was set to <50%, which corresponded to a final output of 2.3 mW for the 488 nm line and 1.2 mW for the 561 nm line. Images were captured every 75 ms beginning 2 s before drug application and continued until the calcium signal decayed. Micro-manager software  was used to acquire images and Image J  and Microsoft Excel  were used for offline analysis. Throughout all of the experiments involving calcium imaging the laser and camera gains were held constant. Thus, it became necessary to exclude those astrocytes, both mutant and wild-type, that had high resting levels of calcium in order to prevent signal saturation. To quantitate calcium signals in astrocytes that qualified for analysis, the entire somatic region was selected as a region of interest (ROI). Then, for each ROI, the time dependent changes in signal intensity were quantitated as ΔF/F2 for 10 m in the dark. The astrocyte was then dialyzed with the loaded cage via the patch pipette along with 0.1 mM Alexa Fluor 488 for at least 10 m. A shuttered, externally mounted fiber optic was used to trigger a 1 s flash from an X-Cite 120 EXFO LED. Control astrocytes were dialyzed with an empty DMNP-EDTA cage and Alexa Fluor 488.For calcium uncaging experiments, 4 mM DMNP-EDTA  was loaded with 3.6 mM CaClFor certain experiments, the efficacy of the uncaging protocol was validated by use of a calcium indicating dye during the uncaging. For this purpose slices were incubated with 10 μM Fluo4FF-AM and astrocytes were loaded with 4 mM DMNP-EDTA caged calcium as previously described. Resting calcium levels were measured for ~2 s using the 488 nm laser line after which the calcium was uncaged using a 1 s flash. At the time of uncaging, it was necessary to close the light path to the camera, limiting the calcium imaging and analysis to the time following termination of the flash.Coronal 80 μm slices were cut from barrel cortex in ice cold 1x PBS, the cortex was removed and transferred to 4% paraformaldehyde (PFA) and fixed for 3 hr at 4°C. Slices were washed with 1x PBS containing 20 mM glycine. Slices were then permeabilized with 1x PBS containing 0.2% triton x-100 for 15 m at 23˚C. Slices were then blocked with 1x PBS containing 3% BSA and 5% normal goat serum for 30 m at 23˚C followed with incubation in the respective primary antibody overnight at 4°C in the PBS-based blocking solution. After overnight incubation, the slices were washed and then incubated in secondary antibody for 2 hr at 23˚C in the PBS-based blocking solution. Following exposure to the secondary antibody, slices were washed and then mounted using Prolong gold with DAPI. Primary antibodies used were: NeuN , GFAP , Par1 . Secondary antibodies used were: Alexa Fluor donkey anti goat-568 , Alexa Fluor donkey anti rabbit-647 , Alexa Fluor donkey anti mouse-488 . All secondary antibodies were purchased from Thermo Fisher .Coronal 80 μm slices were cut from barrel cortex in ice cold 1x PBS, the cortex was removed and immediately lysed in RIPA buffer (25 mM Tris ph 7.6), 150 mM NaCl, 1% NP40, 1% deoxycholate, 0.1%SDS, benzoate nuclease, protease inhibitor). Protein concentration was determined via BCA assay. 40 μg of protein (boiled with loading dye at 70°C for 10 m) was run on a 4–12% bis tris gel with MOPS at 200 V for 1 hr. Gel was transferred to a nitrocellulose membrane using a methanol-based buffer for 1 hr at 100 V. Blot was blocked in 1x TBST with 3% BSA for 1 hr at 23˚C and then incubated in primary antibody with 1x TBST and 1.5% BSA over night at 4°C. Following primary incubation, the blot was washed and then incubated in secondary antibody for 1 hr in 1xTBST with 3% BSA at 23˚C. Blot was then washed before imaging on ODYSSEY. Primary antibodies used were: Par1 , α-tubulin , MeCP2 . Secondary antibodies were conjugated to IR-fluorophore for imaging on Li-Cor Odyssey machine: goat anti mouse- IR680 , goat anti rabit-IR680  donkey anti goat- IR 800 . 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.eLife. Your article has been favorably evaluated by Richard Aldrich (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Astrocytic modulation of excitatory synaptic signaling in a mouse model of Rett syndrome" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This is an interesting study that investigates the impact of MeCP2 and astrocyte gating of neuronal activity in normal and mouse model of Rett syndrome. The authors employ a range of techniques to depolarize astrocytes and measure the frequency and amplitude of events on a neighboring neuron. First, the authors were able to induce the increase in the frequency, not amplitude, of events on neighboring neurons utilizing three techniques  to activate astrocytes. Second, this effect was not directly related to GABA or glutamate release/signaling. Thirdly and most interestingly, the persistent increase was dependent on astrocytic, not neuronal, expression of MeCP2.Overall, the data are compelling and the topic is interesting. Understanding the contribution of astrocytes to neuronal and circuit dysfunction is important. The mosaic model is a powerful way to test cell-type dependence of the phenotype. However, there were also a number of questions and revisions raised by reviewers that should be addressed as outlined below, including the addition of statistics, p values, experimental details to the key figure and results. The authors show that upon non-physiological depolarization of astrocytes, it can impact synaptic activity in surrounding neurons but fail to identify a physiologically-relevant stimuli or potential mechanism. If this is challenging to address experimentally in a timely manner, it is important that the authors include critical and thoughtful discussion of these issues in the revised paper. In addition, some consolidation of Results section and figures is needed to increase clarity and readability for non-experts.Reviewer #1:The manuscript from Rakela and colleagues investigates the impact of MeCP2 and astrocyte gating of neuronal activity in normal and mouse model of Rett syndrome. In this study, the authors employ a range of techniques to depolarize astrocytes and measure the frequency and amplitude of events on a neighboring neuron. First, the authors were able to induce the increase in the frequency, not amplitude, of events on neighboring neurons utilizing three techniques  to activate astrocytes. Second, this effect was not directly related to GABA or glutamate release/signaling. Thirdly and most interestingly, the persistent increase was dependent on astrocytic, not neuronal, expression of MeCP2. I agree with the authors that their work to discriminate the expression of MeCP2 as necessary across the astrocyte/neuronal interaction is important and clever in its execution. While the authors were able to show that GABA/Glutamate signaling was not necessary for persistent changes, the lack of mechanistic insight leaves many questions that should be addressed before publication:1) As indicated in the discussion, Lioy et al. (2011) restored many core features  in a MeCP2-deficient mouse with the selective expression of MeCP2 in only astrocytes. In the study, they showed an increase in the number of vGluT1 terminals and restored morphology. Does the persistent effect shown by Rakela et al. depend on the increase in presynaptic release of vGluT1 synapses? If persistently altered for long periods of time, would it be capable to capture either a morphological or histological change in terminals?2) On a related note, what characteristics of presynaptic release are altered in the face of astrocytic gating? Does the number of release sites increase? The lack of amplitude changes suggests that quantal size is not changing, but how do these changes impact short-term plasticity measures?Reviewer #2:This is an interesting study that reports how astrocyte stimulation affects synaptic response onto pyramidal neurons in control mice and in Rett mouse models. Overall, the data are clear and the topic is interesting and important. I have several suggestions that the authors could accommodate with some revisions.1) For the Par-1 immuno staining, please present some analyses for the expression within astrocytes. Include some average data. The Par-1 response is an important stimulus that is used in this paper, but the reader needs to be convinced that Par-1 receptors exist in cortical astrocytes.- reversal potential is a nice approach used in this study, but the reader needs to be convinced that gramicidin perforated patches behave in the way the authors state. This knowledge is critical to interpret the result in 2) Please provide a clear reference to a published paper showing that gramicidin pores are not permeable to chloride. Again, changing the Cl3) The presentation of the results is a bit long-winded and ponderous. Perhaps sharpen the text to make it briefer and more to the point.+ and or Ca+ in the extracellular fluid? If so, please discuss.4) The authors make the point clearly that they do not know the identity of the substance released from astrocytes to trigger the altered neuronal signaling. If they are sure it is not glutamate release, then they should state this explicitly. Also, could the substance be a change in ATP, K2+ uncaging. As experimental tools these are fine, but the authors should guide the readership by stating in the Discussion that it is likely that all three of these methods are non-physiological and that the conclusions should be interpreted with these thoughts in mind. Thus it is hard to imagine a scenario when astrocytes depolarize to +60mV.5) It is understood that tools to manipulate astrocytes are limited and the authors have had to use strong depolarization, Par-1 agonists and Ca6) Add statistics, p values, n numbers and statistical tests to the key figures so the reader can see what is happening just by looking at the figures.7) Many of the scatter graphs show mean +/- sd. This is fine, but I wonder if it would be better to show box and whisker plots with mean, sd and sem? I suspect some readers will appreciate such graphs more than mean +/- sd.Overall, this study is a valuable addition to the literature and another round of hard work to tighten up the aforementioned aspects will make the study better. I can't comment too much on the mouse model of Rett itself as this is beyond my expertise. However, this aspect will hopefully be addressed by one of the other reviewers.Reviewer #3:This paper examines changes in astrocyte-driven effects on neurotransmission in a mouse model of Rett syndrome. The authors suggest that a mechanism dependent on calcium dependent astrocyte signaling increases the frequency of spontaneous EPSCs and IPSCs. This form of gliotransmission is absent in MeCP2 deficient  mice and shows rescue in WT astrocytes in female mosaic mice. The strength of the paper is in the description of the astrocytic locus of the phenotype. The mosaic model is a powerful way to test cell-type dependence of the phenotype. The weaknesses of the paper revolved around the lack of mechanistic insight and the overinterpretation of experiments.Regardless of the pulse amplitude used for depolarizing stimulation, the physiological relevance of the strong depolarization of astrocytes is highly questionable. It is good that the investigators use a second method of local TFLLR application, but the relevance and specificity of this are also questionable (see below). Were other physiologically-based methods of astrocyte stimulation attempted? Were they unsuccessful?Par1 activation with TFLLR occurs in isolated neurons and causes them to fire action potentials (PMID 21827709). Thus, the possibility of a direct TFLLR on neurons needs to be treated with respect.2+ signal initiated by astrocyte depolarization should be documented in the authors' hands, using the brief-pulse protocols used to elicit the PSC frequency increase. A few papers are cited, but voltage-gated Ca2+ influx in astrocytes is not universally observed.The CaThe authors seem to want to interpret experiments in The attempts to measure EPSCs and IPSCs by clamping midway between reversal potentials appear fraught. Membrane noise is high in 2+ imaging should be shown. How confident can the authors be that images derive from astrocytes? Wouldn't signals from astrocyte processes be most relevant to the studies? How were signals from neurons/neuron dendrites excluded?Figure 12: Raw images that serve as the basis for the Ca2+ signals from neurons and astrocytes are shown, but the authors state that Fluo-AM preferentially labels astrocytes.Supplemental The results seem to suggest that both Ca increase/handling and downstream signal transmission are altered in MeCP2 astrocytes. This is an interesting conclusion but leaves one hanging, without more mechanistic explanation.Other points:Sex of WT mice in initial experiments should be specified.A general description of statistical treatment is given in the Materials and methods. For increased transparency, I would like to see the statistical tests specified in the figure legends for each experiment.Abstract, and perhaps elsewhere: 'astrocyte activation' has a specific connotation that should be avoided in this paper.Subsection “Astrocytes mediate increases in cortical neuron signaling”, second paragraph. Why mention the decay information as data not shown? If this is important to the arguments presented, show the data. If it is not important, omit the reference.Subsection “Astrocytes mediate increases in cortical neuron signaling”, second paragraph. The Results describe astrocyte stimulation as a pulse to +60 mV; the figure legend cites +140 mV.eLife. Your revised article has been favorably evaluated by Richard Aldrich (Senior Editor), a Reviewing Editor, and 1 reviewer.Thank you for resubmitting your work entitled "Astrocytic modulation of excitatory synaptic signaling in a mouse model of Rett syndrome" for further consideration at The manuscript has been improved, rewritten and streamlined as suggested. However, there are a few remaining issues that need to be addressed before acceptance, as outlined below.1) Please clarify and explain rationale for the GABA experiments in 2) Please address remaining concerns related to the design and interpretation of experiments in Reviewer #3:The authors have achieved a more effective and streamlined presentation in this revision. As noted by previous reviews, the main limitation remains the limited mechanistic insight. In addition, some important elements remain unclear.The rationale for the GABA experiments in I cannot overcome my problems with the design of I am still confused about the time course data shown in  Overall, the data are compelling and the topic is interesting. Understanding the contribution of astrocytes to neuronal and circuit dysfunction is important. The mosaic model is a powerful way to test cell-type dependence of the phenotype. However, there were also a number of questions and revisions raised by reviewers that should be addressed as outlined below, including the addition of statistics, p values, experimental details to the key figure and results. The authors show that upon non-physiological depolarization of astrocytes, it can impact synaptic activity in surrounding neurons but fail to identify a physiologically-relevant stimuli or potential mechanism. If this is challenging to address experimentally in a timely manner, it is important that the authors include critical and thoughtful discussion of these issues in the revised paper. In addition, some consolidation of Results section and figures is needed to increase clarity and readability for non-experts.We reduced the number of figure supplements from 7 to 5, added the requested immunohistochemistry figure and further consolidated the primary figures from 13 to 11. We color-coded all recordings, in all of the appropriate figures, to provide easy visual links between the histograms showing the number of events and the corresponding fold changes. We now include statistics and gender within the figure legends for all experimental data. We also shortened and sharpened the Results section. We rewrote the paper to better describe the different forms of stimulation, pointing out directly that depolarization is likely non-physiological and, like so many other studied features of astrocytes, obscures mechanistic insights. We also point out, however, that without our use of depolarization, which acts on one specific astrocyte, we would not have been able to perform the novel experiments in the female mosaic brain  or the tReviewer #1:[…] 1) As indicated in the discussion, Lioy et al. (2011) restored many core features  in a MeCP2-deficient mouse with the selective expression of MeCP2 in only astrocytes. In the study, they showed an increase in the number of vGluT1 terminals and restored morphology. Does the persistent effect shown by Rakela et al. depend on the increase in presynaptic release of vGluT1 synapses? If persistently altered for long periods of time, would it be capable to capture either a morphological or histological change in terminals?Lioy’s findings of morphological changes and elevated vGluT1 levels were based on long-term restoration of MeCP2 in astrocytes in vivo. It is difficult to imagine that similar morphological changes would occur with our astrocyte stimulation that is on the order of tens of seconds, and our resolution in slices limits any detection of fine scale structural changes. Further, our data showing an increase in neurons in the frequency of both GABA and glutamatergic synaptic currents, after astrocyte stimulation, reflects increased release probability and not postsynaptic alterations. We have emphasized this point in the first paragraph of the Discussion. As presynaptic, the increase in glutamatergic responses would therefore reflect release from vGluT1 presynaptic terminals.2) On a related note, what characteristics of presynaptic release are altered in the face of astrocytic gating? Does the number of release sites increase? The lack of amplitude changes suggests that quantal size is not changing, but how do these changes impact short-term plasticity measures?We conclude that the effect is presynaptic and not postsynaptic based on the increase in frequency and absence of change in amplitude for synaptic events, as noted by the reviewer. We stress that in our Discussion. We have no data that address changes in release site number or the mechanisms involved in the short term plasticity as the neuronal circuitry involved is likely complicated.Reviewer #2:[…] 1) For the Par-1 immuno staining, please present some analyses for the expression within astrocytes. Include some average data. The Par-1 response is an important stimulus that is used in this paper, but the reader needs to be convinced that Par-1 receptors exist in cortical astrocytes.To address this important concern we added a new - reversal potential is a nice approach used in this study, but the reader needs to be convinced that gramicidin perforated patches behave in the way the authors state. This knowledge is critical to interpret the result in 2) Please provide a clear reference to a published paper showing that gramicidin pores are not permeable to chloride. Again, changing the ClWe now cite in the Results section the earliest and best studies showing cation selectivity and the lack of permeation by Cl with gramicidin. The references to an excitatory action played by GABA during early development are in the last paragraph of the Discussion.3) The presentation of the results is a bit long-winded and ponderous. Perhaps sharpen the text to make it briefer and more to the point.The overall length changes achieved by consolidation of figures, along with the movement of ponderous pharmacology of basal synaptic currents to supplemental, have resulted in a ‘more to the point’ Results section.+ and or Ca+ in the extracellular fluid? If so, please discuss.4) The authors make the point clearly that they do not know the identity of the substance released from astrocytes to trigger the altered neuronal signaling. If they are sure it is not glutamate release, then they should state this explicitly. Also, could the substance be a change in ATP, KWe embraced this criticism and have added more logic behind the time-locked averaging and the use of inwardly and outwardly directed currents to test glutamate and GABA as substances released. We now make it clear in the Results and Discussion that GABA is ruled out on the basis of bidirectional experiments in 2+ uncaging. As experimental tools these are fine, but the authors should guide the readership by stating in the Discussion that it is likely that all three of these methods are non-physiological and that the conclusions should be interpreted with these thoughts in mind. Thus it is hard to imagine a scenario when astrocytes depolarize to +60mV.5) It is understood that tools to manipulate astrocytes are limited and the authors have had to use strong depolarization, Par-1 agonists and CaAs outlined in our response to reviewer 1, we have responded by adding to the Discussion a section related to the use of depolarization and the resulting lack of mechanistic insights. We also make clear that depolarization and calcium uncaging are non-physiological stimuli, but we do take issue with the Par1 activation being non-physiological. It has a known biological ligand, mimicked by TFLLR, and a well characterized signaling pathway. The claim that Par1 is physiological is now better supported by references in the Results and Discussion.6) Add statistics, p values, n numbers and statistical tests to the key figures so the reader can see what is happening just by looking at the figures.As requested, statistics, statistical tests used, number of recordings, number of animals and gender have been added to every appropriate figure.7) Many of the scatter graphs show mean +/- sd. This is fine, but I wonder if it would be better to show box and whisker plots with mean, sd and sem? I suspect some readers will appreciate such graphs more than mean +/- sd.In some experiments shown the numbers of recordings are not appropriate for SEM and we want to remain consistent throughout the paper with our representation of the results. For this reason, we hope the reviewer will be satisfied with our showing only the mean +/- SD.Reviewer #3:[…] The reviewer raises an important point that we addressed as follows. For every recording, we generated a plot of event number vs. amplitude, similar to the cumulative plot shown in The statement was deleted.Regardless of the pulse amplitude used for depolarizing stimulation, the physiological relevance of the strong depolarization of astrocytes is highly questionable. It is good that the investigators use a second method of local TFLLR application, but the relevance and specificity of this are also questionable (see below). Were other physiologically-based methods of astrocyte stimulation attempted? Were they unsuccessful?TFLLR was the only physiologically relevant stimulation we tried. We chose this agonist because the receptor is highly enriched in astrocytes and the signaling pathway in astrocytes has been characterized. As outlined in our response to the editor and reviewer 2, we have added references to the considerable Discussion supporting a role for TFLLR in a naturally occurring signaling pathway. The specificity of the Par1 receptor for astrocytes is further elaborated in the discussion now on the basis of immunohistochemistry (replicated in our own hands) and specific inhibition of the neuronal response by dialysis of the astrocyte with BAPTA .Par1 activation with TFLLR occurs in isolated neurons and causes them to fire action potentials (PMID 21827709). Thus, the possibility of a direct TFLLR on neurons needs to be treated with respect.Our strongest evidence that TFLLR does not work via neurons is provided by our finding that inclusion of BAPTA in the astrocyte electrode abolishes all activation. Additionally, we detected Par1 by immunohistochemistry in cortical astrocytes but not neurons. We have added a section in the Discussion emphasizing this important issue. We also added to the Discussion a critical evaluation of the paper referenced by the reviewer. That study was restricted to a single class of neurons, shown by the authors to express Par1 on the bases of immunohistochemistry and electrophysiology. However, within that population of neurons, <5% of the cells depolarized in response to TFLLR, and then only after greatly prolonged bath application of >120 sec. The absence of immunohistochemical detection in cortical neurons, the BAPTA block of activation in astrocytes and the differences in time scale for activation between astrocytes and neurons all indicate that Par1 is working through astrocytes in the cortex.2+ signal initiated by astrocyte depolarization should be documented in the authors' hands, using the brief-pulse protocols used to elicit the PSC frequency increase. A few papers are cited, but voltage-gated Ca2+ influx in astrocytes is not universally observed.The CaWe tested for calcium currents using whole cell astrocyte patch clamp recordings. However, the low input resistance and space clamp issue greatly compromised detection. We were also unsuccessful in measuring any effects of depolarization on calcium signals in the soma with calcium-indicating dyes. As noted by this reviewer, the presence of voltage-gated calcium channels remains under dispute, but we believe positive findings from published studies outweigh the negative. To this point, we cite published data showing both T and L type calcium channels in acutely dissociated astrocytes.The authors seem to want to interpret experiments in not block TFLLR activation, then we could rule it out as a potential activator of the neuronal responses, independent of its role in neuronal circuitry. In this way, our results ruled out GABA but not glutamate. The logic and interpretation is now clarified in the Results and Discussion. To further test glutamate, we performed time locked averaging to see if we could detect early synchronized synaptic responses that might reflect direct actions by glutamate .Reviewer #3:The authors have achieved a more effective and streamlined presentation in this revision. As noted by previous reviews, the main limitation remains the limited mechanistic insight. In addition, some important elements remain unclear.The rationale for the GABA experiments in The rationale for the GABA experiments is indeed based on I cannot overcome my problems with the design of We appreciate that separating the inward and outward currents the way we did it is not standard, but it is the only way to perform the experiments in I am still confused about the time course data shown in This reviewer missed the distinction of the paradigm in


HCM is usually linked to higher myofilament Ca2+-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential.The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal CaHIGHLIGHTS- Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable.2+-sensitivity, essential for normal responses to adrenaline.- We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca2+-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca2+-sensitivity.- We have identified a new class of drugs that are capable of both reducing Ca- The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable.- Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers. Troponin, together with tropomyosin and actin constitutes the thin filaments of muscle. In unstimulated muscle contraction is inhibited by the C-terminus of the troponin I subunit binding to actin-tropomyosin and blocking the interaction with myosin crossbridges. Upon stimulation intracellular Ca2+ rises and Ca2+ binds to the troponin C subunit (TnC), the C-terminus of TnI is released from its site on actin and crossbridge cycling can proceed  leading to increased rate and magnitude of contraction. Lusitropy, an increased rate of relaxation allowing for shorter twitch duration, is a key component of adrenergic activation and involves TnI. Phosphorylation of ser22 and 23 of TnI by PKA leads to a 2-fold decrease in Ca2+-sensitivity and a corresponding increase in the rate of Ca2+ release from TnC and is essential for the lusitropic response  and hypertrophic cardiomyopathy (HCM) are often due to single mutations in a protein of the sarcomere and have been extensively studied 2+-sensitivity that is considered to be sufficient to induce the symptoms of HCM. The effect of DCM mutations in contractile proteins on Ca2+-sensitivity is variable with a trend toward deceased Ca2+-sensitivity , compared with similar inactive molecules can provide considerable insight into the mechanism of re-coupling and may lead to the discovery of more potent re-coupling (lead) compounds with therapeutic potential.EGCG and its analogs have been widely studied as Hsp90 inhibitors, so we began our investigations using analogs of EGCG and of Silybin, a natural product with structural similarity, originally studied as Hsp90 inhibitors . To reduce the phosphorylation level (<0.3 mol Pi/mol) troponin was treated with shrimp alkaline phosphatase  and mutant E180G tropomyosin were expressed in a baculovirus/Sf9 system  , tropomyosin (40–60 nM), and troponin (60 nM) to study Ca2+ concentrations to generate Ca2+-activation curves. The data were fitted to the 4-variable Hill equation to yield a value for EC50 and nH. Most assays of the effect of compounds are single measurements. When appropriate EC50 values from replicate experiments were analyzed by paired t-test since the distribution of EC50 has been shown to be normal. Error bars and n values are given in tables and figures.The fraction motile was measured over a range of Ca2+. Most assays were single measurements.For drug screening, motility was measured with paired phosphorylated and unphosphorylated tropomyosin E180G mutant-containing thin filaments at a fixed 0.037 μM Ca2+ activation of contractility using an in vitro motility assay that measures the movement of reconstituted thin filaments over a bed of immobilized myosin heads. A series of measurements, some of which have been previously published . Thus, the effect of EGCG is independent of the mutation that induced uncoupling; in addition, both in man and cat, HCM cases were found where no mutation in sarcomeric genes was detected yet the thin filaments were uncoupled and they could also be re-coupled by EGCG  there were four compounds that were both Hsp90 inhibitors and re-couplers, four that were re-couplers only, four that were Hsp90 inhibitors only and seven that were inactive in both assays  is an example of a compound that is a pure desensitizer as illustrated in Figures r Figure .2+-sensitivity of native thin filaments in either the phosphorylated or unphosphorylated state, in contrast to EGCG  and compound 7 that has a reverse effect differ only by the number and positions of O-methyl groups in the D ring whilst other substitutions in the D ring (compounds 8 and 16) render the compound inactive. In addition, a number of substitutions in the A ring are compatible with recoupling when combined with an appropriate substitution in the D ring   and meta positions.A group of compounds with substitutions containing the core rings A, C, and D of EGCG shift function from good recoupling to no effect and even to reverse re-coupling. The structural differences are small, for instance compound  Figures . Methyla2+-sensitivity. In addition, we have identified a molecular level dysfunction common to both HCM and DCM-causing mutations    Figure . This in2+-sensitivity and unphosphorylated form with high and low Ca2+ sensitivity . Mutations that produce uncoupling can only undergo transition from the unphosphorylated high Ca2+ sensitivity to phosphorylated high Ca2+ sensitivity state and the further transition to low Ca2+ sensitivity is blocked. Dvornikov et al. have proposed structural evidence that a normal phosphorylation-dependent structural transition is blocked in the TnI R145W HCM mutant troponin  could be accounted for if they bound equally to the phosphorylated and unphosphorylated low affinity states. The reverse re-coupler, compound #2+-sensitivity of TnC compatible with our four state model  to troponin subunits have been made, most studies have involved binary complexes (TnI and TnC) and partial sequences  or chromanone (in Silybin) are fundamental. The way in which the substitutions define the structure, charge distribution, hydrogen bonding potential or acidity of the A ring, apparently involved in its intercalation between TnI and TnC, is likely to be critical for the compound effect. In the EGCG analogs it seems that either the B ring  or the D ring  are sufficient to confer re-coupling activity, so one ring may be able to assume the configuration of the other when one of them is absent. In Silybin and Dehydrosilybin, the lignan part is not fully needed for function  and only the B isomers are recouplers, whilst the A stereoisomers of Silybin and Dehydrosilybin are inactive as recouplers. In this case it seems likely that the A isomers have an unfavorable conformation that prevents the A and D rings binding in the same way as the B isoform. It is noteworthy that the B isomer is more active than the A isomer is several different bio-assays (Kren and Walterová, 2+ sensitivity by TnI phosphorylation is the loss of the lusitropic response to beta adrenergic stimulation, which is usually accompanied by blunting of the whole physiological adrenergic response. This is demonstrated most directly by a lack of response to dobutamine stimulation observed at the whole animal, papillary muscle and single cardiomyocyte levels (Song et al., The main effect of uncoupling of the modulation of CaAS, AM, MP, AC performed the experiments reported here; BB, AK, VK, DB prepared and supplied EGCG and silybin analogs; SM obtained funding, directed research and wrote the paper. All the authors contributed to editing and completing the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.