Peripheral artery disease (PAD) is caused by atherosclerotic occlusions in the legs. It affects approximately 8-12 million people in the United States alone, one-third of whom suffer from intermittent claudication (IC), defined as ischemic leg pain that occurs with walking and improves with rest. Patients with IC suffer a markedly impaired quality of life and a high perception of disability. Improving pain-free walking time is a primary goal of rehabilitation in this population.2peak) during a maximal-graded cardiopulmonary exercise test (max CPX) and (2) functional capacity: 6-minute walk (6MW) distance. The secondary aims will provide mechanistic insights into the exercise outcome measures and will include (1) gastrocnemius muscle oxygenation during exercise via near-infrared spectroscopy (NIRS); (2) gastrocnemius muscle angiogenesis: capillaries per unit area and per muscle fiber, and relative fraction of type I, IIa, IIb, and IId/x fibers; and (3) vascular health/function via brachial artery flow-mediated dilation, lower-limb blood flow via plethysmography, and pulse wave velocity and reflection.The nitric oxide (NO)-PAD trial is designed to compare the effects that 12 weeks of supervised exercise training, in combination with a high inorganic nitrate-content (beetroot [BR] juice) beverage or placebo (PL) beverage, has on clinical outcomes of exercise and functional capacity in two groups of PAD+IC patients: exercise training plus beetroot (EX+BR) and exercise training plus placebo (EX+PL). The primary aims of this randomized controlled, double-blind pilot study are to determine group differences following 12 weeks of EX+BR versus EX+PL in the changes for (1) exercise capacity: pain-free walking time , peak walk time (PWT), and maximal exercise capacity . They will be randomized to either the EX+BR or EX+PL group where participants will consume a beverage high in inorganic nitrate (4.2 mmol) or a low-nitrate placebo, respectively, 3 hours prior to each training session.d effect sizes of medium to large.Data collection from this study has been completed and is in the process of analysis and write-up. While the study is too underpowered—EX+BR, n=11; EX+PL, n=13—to determine between-group differences in the primary outcomes of COT, PWT, and 6MW, preliminary observations are promising with Cohen Exercise training is currently the most effective therapy to increase functional capacity in PAD+IC. If the addition of inorganic nitrate to an exercise regimen elicits greater benefits, it may redefine the current standard of care for PAD+IC.ClinicalTrials.gov NCT01684930; https://clinicaltrials.gov/ct2/show/NCT01684930 (Archived by WebCite at http://www.webcitation.org/6raXFyEcP) Peripheral artery disease (PAD) is caused by stenosis of the arteries in the lower limbs, leading to a reduction in blood flow to the legs . It affeThus far, interventions aimed at improving clinical outcomes in patients with PAD have been either surgical, pharmacological, or exercise based, with supervised exercise training identified as the current best intervention for improving functional outcomes such as peak walking time and pain-free walking time -7. ExercDysfunction of the vascular endothelium is a hallmark of cardiovascular diseases, including PAD . A key f2-), while once considered to be a biologically inert marker of NO, has recently been identified as an alternative NO source that can be reduced to NO under low oxygen conditions, such as hypoxia or ischemia ) will experience a greater clinical benefit in pain-free walking time  and PWT than those undertaking exercise and a placebo beverage (exercise training plus placebo juice [EX+PL]) alone. Specifically, the primary aim of this study is to determine group differences (EX+BR vs EX+PL) in the changes in the following:2peak) during a maximal-graded cardiopulmonary exercise test (max CPX).Exercise capacity: pain-free walking time (COT), PWT, and maximal exercise capacity/peak oxygen uptake .Gastrocnemius muscle angiogenesis: capillaries per unit area and per muscle fiber, and relative fraction of type I, IIa, IIb, and IId/x fibers.Vascular health/function via brachial artery flow-mediated dilation, lower-limb blood flow via plethysmography, and pulse wave velocity (PWV) and reflection.The NO-PAD trial is a pilot, randomized, double-blind, per-protocol design with four different assessment time points see . This stThe first round of assessment will act as a baseline with no exercise intervention or supplementation. Following this, all participants will be randomized to groups consuming either a 70 mL (4.2 mmol nitrate) beetroot juice  or an identical nitrate-depleted placebo. Participants will then repeat the baseline assessments, except for the muscle biopsy, but will consume their assigned beverage 3 hours prior to each visit.Participants will continue consuming their beverage 3 hours prior to each of their 36 exercise training sessions. Periodically, subjects will be selected for unannounced blood draws at training visits to check plasma nitrate and nitrite concentrations. Post-exercise training, participants will complete the same assessments as the baseline visit while still acutely consuming their beverage before visits. Both groups will then stop consuming the beverage for at least 1 week prior to the final visit. The half-life of nitrate is 5-8 hours and as the supplementation protocol is three acute doses per week, a 1-week washout period should be sufficient to minimize any possible residual effect of the nitrate . To maxiWe aim to recruit a total of 30 participants—equal numbers of men and women—between the ages of 40 and 80 years with diagnosed PAD from the clinics and community at Duke University Medical Center. We anticipate a dropout rate of approximately 15%-20% and, therefore, 24 patients out of the 30  are expected to complete the study. This study will have continuous rolling enrollment until the recruitment goals are met. Subjects will be recruited primarily through collaboration with Duke University Medical Center cardiovascular physicians and vascular surgeons. Clinic medical records will be reviewed to prescreen patients for inclusion and exclusion criteria see .Inclusion criteria:1. Aged between 40 and 80 years.2. Diagnosed peripheral artery disease  with intermittent claudication.3. No major changes in medications for at least 3 months.Exclusion criteria:1. Foot ulcers, advanced neuropathy, gangrene, or other musculoskeletal condition that could limit exercise performance.2. Type 1 diabetes or glycated hemoglobin >8.5%.3. A major cardiovascular event within the previous 6 weeks or a planned hospitalization within the next 2 months.4. Any cardiovascular condition that impacts safety of completing a cardiopulmonary exercise test, including history of significant left main or three-vessel coronary artery disease (>70% stenosis), recent myocardial infarction (6 weeks), chest pain during cardiopulmonary exercise test, or >2 mm ST depression during exercise; foot ulcers/advanced neuropathy or other musculoskeletal condition that could limit exercise performance.5. Allergy to beets or proton pump inhibitors.6. Refusal or inability to abstain from the use of proton pump inhibitors for 24 hours prior to testing.Records of those criteria will be noted and the appropriate medical doctor will introduce the study to the patient. Patients who consent to being contacted by the research team will undergo a telephone screening questionnaire to determine if the study eligibility criteria are met. Eligible and interested patients will then attend an orientation visit and a consent meeting. During this initial visit, the study details will be explained verbally and the individuals will have the opportunity to read over the approved informed consent document and ask any questions they may have about the trial. The patients can then choose to sign the informed consent form or decline participation. Following the consent visit, enrolled subjects will return on a separate day to undergo a physical examination and a review of their medical history and medications by the study physician. Participants will then be scheduled for their baseline assessment visits. Details of the testing occurring at each time point is outlined in Following initial screening and baseline testing, subjects will be randomized to consume either a high-nitrate-containing beetroot beverage or a low-nitrate placebo throughout the duration of the exercise training. Randomization should ensure the groups are initially equal within either the EX+BR or EX+PL intervention. Both groups will be instructed to consume one 70 mL bottle of beetroot juice (containing 4.2 mmol inorganic nitrate) 2.5-3 hours prior to every testing visit and training session between time point 2 and time point 3. The beetroot juice bottles will be identical in appearance and taste. The bottles will be color coded by a third party to ensure that the research staff remain blinded while providing the correct supplement to each participant.Scheduled but unannounced blood draws will be performed throughout the study to confirm participants are conforming to their appropriate beverage allocations.All subjects will attend supervised exercise training sessions three times per week for a total of 12 weeks at the Duke Health and Fitness Center at the Duke University Medical Center for Living. Each session is structured to last approximately 45-60 minutes, including taking vital signs—heart rate and blood pressure (BP)—before and after the training session. To meet minimum exercise session attendance requirements, participants will need to complete 34 of the 36 exercise training sessions and should not miss more than 1 week of training  consecutively. Additional sessions will be added if necessary to ensure that an adequate exercise stimulus is given to each subject so that any decreases in fitness or function during unintended breaks are mitigated. The study will be analyzed under per-protocol criteria. Subjects who withdraw from the study prior to completion will be excluded from the final analysis. However, attrition rates will be reported and documented.Participants will not be excluded due to current exercise habits, but all participants will be instructed to maintain their normal activity levels throughout the exercise intervention.The exercise session will consist of approximately 5 minutes of warm-up, 30 minutes of accumulated exercise (not including rest periods), and cooldown (if necessary). The walking treadmill exercise prescription is individualized to each participant’s exercise capacity during baseline testing and their rate of progression. The protocol requires participants to accumulate a total of 30 minutes of walking with claudication pain. Neither rest time nor time spent exercising before pain onset will count toward the 30-minute goal. The patient will be encouraged to continue walking as far as they can, but will self-select when they require a rest break. Exercise and rest periods are repeated during each session until the minimum total of 30 minutes of walking time is reached each session. When a patient can walk 8-10 minutes at the initial workload, the grade will be increased by 0.5%, or the speed increased by 0.1 mph as tolerated, to ensure workload progression.Subjects will be supervised by a trained exercise physiologist and will have continuous telemetry heart rate monitoring using the Polar heart rate monitor (Polar Electro). Fatigue will be assessed through rating of perceived exertion via the Borg 6-20 scale; blood pressure will be checked both before and after exercise as well as periodically during each exercise.Each exercise training session will be documented with preexercise and postexercise vital signs, time of day beverage was consumed, total exercise time, and workload  recorded. Body weight and dietary intake will be assessed on a weekly basis. All research team members working with the subjects will be blinded to the treatment intervention and every attempt will be made keep encouragement and workload adjustment standardized between subjects.The subjects and research staff conducting the testing will be unaware of group assignments. The primary investigator will know to which treatment group the subjects are randomized, since changes in plasma nitrate and nitrite will be used to determine beverage volumes and tolerance. Because of the potential for bias, however, the primary investigator will not be directly involved in collecting or analyzing any of the exercise performance or training data.For all testing visits, participants will be advised to avoid exercise and consuming alcohol for 24 hours prior to the examination day. They will also be asked to avoid tobacco and caffeine for 3 hours prior to any testing or exercise visit.The maximal cardiopulmonary exercise test (CPX) with a 12-lead electrocardiogram and expired gas analysis will be conducted using a modified Gardner protocol. This protocol starts at 2.0 mph and 0.0% grade and increases by 2.0% grade every 2 minutes at each stage .Prior to commencing the protocol, participants will be familiarized with the Borg rating of perceived exertion scale as well as the claudication pain scale. Resting measures of gastrocnemius tissue oxygenation, via NIRS, and oxygen consumption, via a metabolic cart, will be recorded.2peak, rather than a maximum.Throughout the test, heart rate, blood pressure, NIRS, and rating of perceived exertion will be monitored. Assuming there are no adverse events, participants will walk on the treadmill until volitional exhaustion. These participants will likely be limited by leg claudication pain and, therefore, we expect to attain a VO2peak will be the average of the last 30 seconds of exercise.The measure of pain-free walking time will be recorded as the total time walked prior to the onset of claudication pain. During the max CPX, participants will be instructed to inform a research team member when they first feel pain in their leg during exercise—this will be recorded as COT. PWT will be recorded as the total time (in seconds) that the participant walked on the treadmill. VOPrior to beginning the max CPX, a 20-gauge intravenous catheter will be placed in an antecubital vein. Approximately 5 mL of blood will be taken prior to the max CPX testing (Pre) and at 10 minutes following exercise termination (Post). Samples will then be separated into five 1 mL Eppendorf tubes containing 5 µL heparin (1-1000 U/mL) and centrifuged at 5000 g for 3-4 minutes . AfterwaAll NO metabolite concentrations will be measured within 30 minutes of defrosting by chemiluminescence using the NOA 280 nitric oxide analyzer (Sievers Instruments) as per the manufacturer’s instructions. The reductant used for nitrite analysis will be potassium iodide in acetic acid, which has the reduction potential to convert nitrite to NO; this reductant is insufficient to reduce any higher oxides of nitrogen such as nitrate and, thus, is relatively specific for nitrite. To obtain concentrations of total plasma nitrogen oxides, we will use the same apparatus with a stronger reductant, vanadium chloride in 1 M HCl, at 94°C. This reduces the sum of all nitrogen oxides with an oxidation state of +2 or higher, which is predominantly nitrate (µM), but also includes both nitrite (nM) and nitrosothiols (nM).NIRS has been shown to be a reproducible and reliable method to determine tissue oxygen stores in human skeletal muscle tissue . The NIRThe tissue oxygenation will be assessed utilizing the NIRS device to obtain an index of fractional oxygen extraction—[deoxy(Hb + myoglobin)]—and percent oxygen saturation at rest at the beginning of the exercise test and continuously monitored throughout the max CPX and recovery period .For the vascular testing visit, subjects will be instructed to withhold all medications and consume no food for 8 hours prior to the exam. All vascular testing will be performed between 8 am and 11 am in a temperature-controlled room following 10-15 minutes of quiet relaxation.Endothelial dysfunction is a precursor to the development of atherosclerosis and an independent predictor of cardiovascular events and clinical outcomes . BrachiaAbsolute and relative changes in brachial artery diameter = (peak posthyperemia diastolic diameter − baseline diastolic diameter)/baseline diastolic diameter × 100 (1)Central blood pressures—the pressures by which the internal organs are perfused—have been shown to be strongly related to clinical outcomes . A speciTo capture PWV, sequential measurements of arterial pressure waves will be taken at the carotid artery, using applanation tonometry, and femoral artery, using a specialized thigh cuff. The surface distances from the sternal notch to the carotid and femoral sites will be measured and input into the software for calculation of the PWV. Pressure wave transit times to each site will be measured using the foot-of-the-wave method:The ankle-brachial index (ABI) is the ratio of blood pressure in the feet in comparison to the arms and is linked with mortality and morbidity rates ,48. MeasFollowing the ABI procedure, the participant will be kept supine throughout the plethysmography measurements. Lower-limb blood flow will be assessed both at rest and following reactive hyperemia via the use of a Hokanson A16 mercury-in-silastic strain gauge plethysmograph (Hokanson Inc). Initial setup requires the patient to lie supine, with the legs elevated slightly above the level of the heart to facilitate venous emptying. Two BP cuffs will be placed on the upper thigh of the leg to be assessed: one will be used for arterial occlusion and one for venous occlusion. A mercury-in-silastic strain gauge will be placed around the widest part of the calf to allow for the indirect measurement of blood flow via changes in lower-limb diameter. Resting measures will be acquired by inflating and deflating the venous occlusion thigh cuffs to approximately 50 mmHg  every 10 seconds for five cycles. During inflation, the mercury-in-silastic strain gauge will be placed under stress, which will then be graphically represented as a pressure response curve on the Hokanson A16 machine. The average of three resting measures will be used as the baseline arterial inflow measure. Following 5 minutes of rest, the thigh cuff will be inflated to 30 mmHg above systolic pressure to induce arterial occlusion for 5 minutes. Upon release of the occlusion cuff, the same procedure used for resting measures will be initiated whereby the venous occlusion cuff will be inflated and deflated to create a pressure response curve for blood flow following reactive hyperemia .Prior to muscle biopsies, all subjects will be asked to refrain from anticoagulant medications. Samples from the gastrocnemius muscle will be obtained using the Bergstrom percutaneous needle biopsy technique. Biopsy sites will be anesthetized with a 2% Xylocaine solution and a 0.5 cm incision will be made through the skin and fascia. Separate samples will be taken with approximately five passes of the needle into the muscle. All samples will then be prepared immediately by weighing and then dividing the samples for later analysis. A tissue sample of approximately 100 mg is consistently obtained with a triple pass from a single insertion; we have demonstrated that this amount of tissue is sufficient for analyzing muscle fiber composition and angiogenesis. Visible blood and connective tissue will be removed and the specimens divided longitudinally. Portions for RNA (40 mg) analyses will be frozen in liquid nitrogen and stored at -70°C. Another portion to be used for histochemical analysis (~30 mg) will be oriented such that the fibers run longitudinally, mounted on cork-embedding medium (OCT compound), and frozen in isopentane cooled with liquid nitrogen.For the analysis of the muscle tissue, after thawing the tissue, measurements will be taken for markers of angiogenesis including the following: capillaries per unit area and per muscle fiber, endothelial cells with surrounding pericytes, and relative fraction of type I, IIa, IIb, and IId/x fibers. Additionally, oxidative capacity of the fibers will be quantified via mitochondrial volume with citrate synthase activity.Informed consent has been obtained from each patient and the study has been approved by the Duke University School of Medicine Institutional Review Board. The study is being run under an Investigational New Drug application filed with the US Food and Drug Administration.This is a repeated-measures design, with the purpose of assessing change over time for the two intervention groups. Since this is a pilot study, the overarching goal of the analyses is to derive effect sizes  for this intervention for a future larger, confirmatory study. We will conduct the analyses chronologically in three phases: (1) descriptive analyses of dependent and independent variables; (2) bivariate analyses of the association between group membership and the outcome measures; and (3) controlled multivariable analyses, which assess the association between groups and the outcomes controlling for important covariates. Randomization should ensure the groups are initially equal; however, we will control for important baseline variables, including the two outcomes of interest. We will not control for any variables observed postrandomization . Our genit is the change in the outcome over the time points, t is an indicator of change period  depending on the wave of measurement, and i is an indicator of the individual . β1 is an indicator variable indicating group membership. β2 is an indicator variable indicating the time of testing: baseline, baseline on supplement, postintervention on supplement, and postintervention off supplement. β3 is a Group × Timet interaction. As listed, β4 is a vector of regression weights linking the design matrix of covariates—time varying and time invariant—to the outcome of interest. We will center Timet=0 at the final 12-week time point, which will allow us to test if there are differences at the end of the trial, β1=0. Follow-up analyses will assess the impact of Group × Timet interaction and the functional form of the change over time. Inclusion of covariates into the model, including baseline measures, may add precision to the estimates and allow us to assess the generalizability of the effects across the covariates. The list of covariates  will be developed prior to any analysis. This is a small sample (n=24); hence, the number of covariates we can utilize in any regression is minimal: ≈10 cases per variable [P=.10), it will be retained in models with Group, Timet, and Group × Timet (see above).where Yvariable . We willThis is a pilot study and we do not expect to declare statistical significance for any of the variables assessed under this analytic structure. However, if the target 12 subjects per group is attained—EX+BR and EX+PL; 24 total subjects—and if a power of 80% with alpha set to .05 (two-tailed) is used, then we will be able to detect a 0.826 standardized difference in the difference in the change labeled as “large” in the statistical power literature . We willAs noted previously, the results from this pilot study will be used to power a subsequent, larger, adequately powered confirmatory clinical trial of the impact of beetroot juice on the outcomes listed above. The results, along with theory and clinical significance, will guide the choice of the primary outcome(s) for that trial, with appropriate control for type I error. While the results of this pilot study will prove useful in the calculation of power, we are aware of the many risks of “playing the winner” in choosing to power a subsequent trial from pilot study results only ,51.d effect sizes of medium to large.Data collection from this study has been completed and is in the process of analysis and write-up. While the study is too underpowered—EX+BR, n=11; EX+PL, n=13—to determine between-group differences in the primary outcomes of COT, PWT, and 6MW, preliminary observations are promising with Cohen Improving pain-free walking and exercise capacity are key goals for the treatment and rehabilitation of patients with peripheral arterial disease . This stThe physiological basis for this trial relies on the fact that supplementation with inorganic nitrate will lead to increased plasma nitrite that can be transported throughout the circulation and is reduced to NO in tissue with a low partial pressure of oxygen. This hypoxic tissue-targeting effect may be particularly pertinent for patients with PAD+IC experiencing tissue ischemia during exercise participation. While it is well established that consuming inorganic nitrate increases circulating levels of plasma nitrite ,52, reseThere are three mechanisms that lend support to nitrate supplementations’ clinical utility in improving exercise performance in patients with PAD. First, acute supplementation data suggest that increasing plasma nitrite via beetroot juice improves ischemic tissue perfusion leading to increases in pain-free walking time and aerobic function . Second,The small sample size and the focus on patients with PAD+IC only limit the ability to broadly apply the results of the study to the larger PAD population and to generalize these results to subsequent studies. This study also uses a per-protocol design with the intent that data may provide the physiological proof of concept for the intervention and allow us to derive variance data and effect sizes within and between groups to inform future studies, which will utilize an intent-to-treat criterion in the analytic structure.This study has been designed to investigate a new therapeutic approach for the treatment of PAD+IC. If successful, the results could influence current medical practice for these patients, while also providing mechanistic insights into potential physiologic targets for other interventions. Research clearly identifies supervised exercise training as being one of the most effective interventions for improving exercise tolerance in patients with PAD ,53,54. W


In many excitatory synapses, mobile zinc is found within glutamatergic vesicles and is coreleased with glutamate. Ex vivo studies established that synaptically released (synaptic) zinc inhibits excitatory neurotransmission at lower frequencies of synaptic activity but enhances steady state synaptic responses during higher frequencies of activity. However, it remains unknown how synaptic zinc affects neuronal processing in vivo. Here, we imaged the sound-evoked neuronal activity of the primary auditory cortex in awake mice. We discovered that synaptic zinc enhanced the gain of sound-evoked responses in CaMKII-expressing principal neurons, but it reduced the gain of parvalbumin- and somatostatin-expressing interneurons. This modulation was sound intensity-dependent and, in part, NMDA receptor-independent. By establishing a previously unknown link between synaptic zinc and gain control of auditory cortical processing, our findings advance understanding about cortical synaptic mechanisms and create a new framework for approaching and interpreting the role of the auditory cortex in sound processing. Many people find it easy to follow a conversation while on a busy city street, but this seemingly simple task requires sophisticated processing of sounds. The brain must accurately distinguish speech sounds from background noise, even though the volumes and pitches of those sounds overlap. To make this possible, neurons that process sounds continually adjust the relationship between the volume of a sound and the size of their response. This helps the brain to distinguish more precisely between different sounds, but how this works remains unclear.Zinc ions form part of almost 3,000 different enzymes and regulatory proteins, and also help neurons to communicate with one another at junctions called synapses. Changes to the amount of zinc ions at the synapses have been seen in disorders including depression and Alzheimer’s disease. By imaging the brains of mice, Anderson, Kumar et al. now show that zinc ions affect how the healthy brain processes sounds.Treating the mice with a substance that temporarily mops up zinc ions changed how neurons responded to sounds of different volumes. This revealed that zinc ions cause excitatory neurons, which activate neighboring cells, to increase their responses to sounds. Conversely, zinc ions cause inhibitory neurons, which reduce the activity of other cells, to decrease their responses to sounds. The overall effect is to change the balance of excitatory and inhibitory activity in areas of the brain that process sound. Anderson, Kumar et al. propose that these changes make it easier for the brain to process and distinguish different sounds as the environment changes from quiet to loud and vice versa.As well as revealing a role for zinc ions in normal hearing, these findings may help us to understand disorders such as tinnitus and auditory neuropathies . Both tinnitus and auditory neuropathies involve changes in the brain’s ability to increase or decrease its responses to sounds with particular characteristics – processes that may involve the activity of zinc ions. As a constituent for nearly 3000 proteins, zinc plays a key role in protein structure, enzymatic catalysis, and cellular regulation . AlthougEx vivo studies established that synaptic zinc is an inhibitory neuromodulator of AMPA receptors, NMDA receptors, and vesicular release probability at lower frequencies of synaptic activity . In contTo answer this question, we investigated the role of synaptic zinc in shaping the sound-evoked activity in cortical neurons of awake mice. Namely, we investigated whether synaptic zinc modulates the relationship between sound intensity and the amplitude of neuronal evoked responses – termed gain modulation. Gain modulation controls the dynamic range of neuronal responses to sounds and is a fundamental feature of sound processing enabling adaptation to changes in sound stimulus statistics, as well as central compensation to peripheral damage . We usedWe began our exploration by visualizing sound-evoked responses in the auditory cortex of awake mice. To image and quantify the cortical sound-evoked responses, we used adeno-associated virus (AAV) driven by the synapsin promoter to express the genetically-encoded calcium indicator GCaMP6s in auditory cortical neurons  promoter to express GCaMP6f in principal neurons . Linear To track the origin of the extracellular zinc increasing the gain of principal neurons, we studied the effects of ZX1 in ZnT3 KO mice, which had been injected with AAV-CaMKII-GCaMP6f. ZX1 had no effect on the sound-evoked responses of principal neurons in ZnT3 KO mice, indicating that synaptic zinc increases the gain of principal neurons .Whereas synaptic zinc is, mostly, an inhibitor of glutamatergic neurotransmission , zinc chTo image and quantify the sound-evoked responses of PV neurons, we injected AAV expressing Cre-dependent GCaMP6f  into the auditory cortex of PV-Cre mice . Linear In PV neurons, the enhancing effect of ZX1 on sound-evoked responses was largest for sounds of 60–80 dB SPL  – the inWe next focused on the effect of zinc chelation on the somatostatin-expressing interneurons (SOM neurons), which inhibit PV neurons . We seleThe smaller ZX1-induced enhancement of the sound-evoked responses of PV neurons to lower intensity sounds (30–40 dB SPL) is consistent with the ZX1-induced increases of the sound-evoked responses of SOM neurons to the same sound intensities . TogetheWe next investigated the effect of zinc chelation on the vasoactive intestinal polypeptide-expressing interneurons (VIP neurons), which inhibit SOM and PV neurons . We seleWe next focused on the molecular targets  that contribute to the effects of synaptic zinc on cortical sound-evoked activity. Because nanomolar levels of zinc inhibit NMDA receptors NMDARs; , we testThe ZX1-induced increases in the sound-evoked responses of PV neurons in the presence of APV  were notNext, we investigated whether NMDARs contribute to the effects of synaptic zinc on the sound-evoked responses of SOM neurons. APV reduced the sound-evoked responses of SOM neurons  and abolWe next tested whether NMDARs contribute to the effects of synaptic zinc on the sound-evoked responses of VIP neurons. APV enhanced the sound-evoked responses of VIP neurons  and abolTranscranial imaging reflects a sound-evoked population fluorescent signal arising from neurons residing in different cortical layers, as well as from different neuronal compartments (e.g. somata vs. dendrites). To explore the layer and sub-cellular specificity of the zinc-mediated effects on gain control, we next performed two-photon imaging of individual A1 L2/3 neurons in awake mice. In these experiments, we investigated the effects of ZX1 on the sound evoked responses from individual L2/3 neuronal somata (Methods). We began these experiments by injecting AAV-CaMKII-GCaMP6f into A1 to record responses from principal neurons . After locating A1 as shown in We next measured the effects of ZX1 on the responses of individual L2/3 PV neurons. We injected AAV-Flex-GCaMP6f into PV-Cre mice and performed 2-photon imaging . We measIn individual L2/3 PV neurons, in contrast to our transcranial measurements, we did not observe any increases in their somatic responses to sounds of 30 dB SPL. Since calcium transients in neuronal dendrites can be tuned to different stimulus features compared to somata , and canWe performed similar two-photon experiments in individual L2/3 SOM and VIP neurons  and the Our results show that synaptic zinc modulates the gain of sound-evoked auditory cortical responses. In the auditory system, as sound statistics of the auditory environment change, auditory neurons adjust their gain – the dynamic range of their responses – to match the stimulus statistics. In the auditory nerve, inferior colliculus, and auditory cortex, neurons adapt their responses to match the mean sound intensity levels . In addiSensory experience alters synaptic zinc levels in primary somatosensory and visual cortex ; howeverSynaptic zinc is present in many cortical areas  and dynaGain modulation is mediated by selective increases or decreases in cortical inhibition . Recent The lack of ZX1 effects on the A1 neuronal responses to specific sound intensities may reflect circuit-based cancelation of opposing ZX1 effects. Although the bulk application of ZX1 limits our ability to distinguish cell- from circuit-specific effects, the lack of ZX1 effect on the sound-evoked responses of principal and VIP neurons to lower sound intensities (30–50 dB SPL), and on the responses of SOM neurons to higher intensities (70–80 dB SPL) suggests that the disruption of extracellular zinc signaling does not affect the balance of excitation and inhibition in these cases . In prinBlockade of NMDARs with APV reduced the sound-evoked responses of PV neurons  and incrThe responses of PV neurons to sound are the most sensitive to zinc modulation, both in amplitude and sound intensity span . It is iWe observed both NMDAR-dependent and NMDAR-independent zinc-mediated effects on A1 responses to sound. This is consistent with in vitro studies showing that synaptic zinc inhibits, albeit at different concentrations, NMDARs , AMPA reIn the presence of APV, the lack of ZX1 effects on the sound-evoked responses of SOM and VIP neurons may be due to potential circuit- or to direct but multiple target-based cancelation of ZX1 effects, and it therefore does not necessarily support total NMDAR-dependence. Despite these limitations in the interpretation of the APV experiments showing negative results, our APV experiments showing enhancing effects of ZX1 on the sound-evoked responses of principal and PV neurons, suggest that synaptic zinc exerts its effects on these neurons, at least partially, in an NMDAR-independent manner. Nonetheless, future experiments investigating the effect of sound-evoked zinc on subthreshold conductances mediating the sound-evoked response of different types of neurons in A1; in vitro experiments on the effect of synaptic zinc on NMDAR and AMPAR EPSCs in A1 synapses; as well as the use of optogenetic approaches are needed to further resolve the detailed molecular mechanisms via which synaptic zinc shapes the sound-evoked responses of A1 L2/3 neurons.Our findings are mostly specific to cells and circuits in L2/3. In the neocortex, synaptic zinc containing terminals are found predominantly in L1, 2/3 and 5, with moderate presence in L6 and light presence in L4 . This diTogether, our results and previous findings on the role of zinc in modulating glutamatergic neurotransmission demonstrate that synaptic zinc fine-tunes excitatory synaptic transmission and sensory processing. Although this fine-tuning does not impair baseline neurotransmission or basic sensory and sensorimotor functions, such as hearing thresholds and prepulse inhibition , it has Slc30a3) KO and WT mice , were used for experiments shown in Pvalb)-Cre mice  were used for experiments shown in Sst)-Cre mice  were used for experiments shown in All procedures were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Male and female ZnT3  19 and P36 were anesthetized with inhaled isoflurane  and secured in a stereotaxic frame . Core body temperature was maintained at ~37°C with a heating pad and eyes were protected with ophthalmic ointment. Lidocaine (1%) was injected under the scalp and an incision was made into the skin at the midline to expose the skull. Using a 27-gauge needle as a scalpel, a small craniotomy (~0.4 mm diameter) was made over the temporal cortex . A glass micropipette, containing AAV vectors, driven by synapsin promoter for neuron-specific expression, was inserted into the cortex 1 mm past the surface of the skull with a micromanipulator (Kopf). We used AAV9.CaMKII.GCaMP6fast.WPRE.SV40 (GCaMP6f) and AAV9.CAG.Flex.GCaMP6f.WPRE.SV40 for two-photon imaging and AAV9.Syn.GCaMP6slow.WPRE.SV40 (GCaMP6s), AAV9.Syn.GCaMP6f.WPRE.SV40, AAV9.CaMKII.GCaMP6f.WPRE.SV40, and AAV9.CAG.Flex.GCaMP6f.WPRE.SV40 for transcranial imaging experiments . The gl2, 1.3 MgCl2, 20 NaHCO3, 3 Hepes, and 10 D-glucose, pH = 7.25–7.35, ∼300 mOsm. For better optical access of the auditory cortex, we injected lidocaine-epinephrine  into the temporal muscle and retracted a small portion of the muscle from the skull. Mice were then positioned under the microscope objective in a sound- and light-attenuation chamber containing the microscope and a calibrated speaker . Acoustic stimuli were calibrated with ¼ inch microphone  placed at the location of the animal’s ear within the chamber.11–24 days after AAV injections, mice were prepared for in vivo calcium imaging. Mice were anesthetized with inhaled isoflurane  and positioned into a head holder. Core body temperature was maintained at ~37°C with a heating pad and eyes were protected with ophthalmic ointment. Lidocaine (1%) was injected under the scalp and an incision (~1.5 cm long) was made into the skin over the right temporal cortex. The head of the mouse was rotated ~45 degrees in the coronal plane to align the pial surface of the right temporal cortex with the imaging plane of the upright microscope optics. The skull of the mouse was secured to the head holder using dental acrylic  and cyanoacrylate adhesive. A tube  was placed around the animal’s body to reduce movement, and the mouse received an injection of the sedative chlorprothixene (0.36 mg/kg intramuscular) to reduce animal movement during in vivo imaging . A denta2 of the image) at a frame rate of 20 Hz. After locating A1 in each animal , we reanesthetized the mouse with isoflurane and performed a small craniotomy (0.4–1 mm2) adjacent to the location of A1 . Using a micromanipulator , we inserted a glass micropipette backfilled with mineral oil and connected to a 5 μL glass syringe into the cortex as above. The pipette contained ACSF including 100 μM of ZX1 and 50 μM Alexa-594. Once the pipette was inserted into the cortex, we removed the isoflurane. After ten minutes of recovery from isoflurane, we presented sound stimuli  while measuring the changes in GCaMP6 fluorescence. After recording the responses to different sounds (3 to 5 presentations of each sound level), we began to infuse the ZX1 solution into the cortex at a rate of 30 nL/min. We monitored the intracortical spread of the ZX1 solution with red epifluorescence optics  and a green LED  for transcranial illumination. After ten to twenty minutes, we observed strong transcranial red fluorescence throughout A1 indicating that the ZX1 solution was present in the cortex. At this point we reduced the pump speed to 9 nL/min and remeasured the sound-evoked responses. For experiments in which we infused 1 mM APV and then 1 mM APV and 100 μM ZX1 into the cortex, we constructed two-barrel infusion pipettes by hot-gluing 2 infusion pipettes together so that their tips were within ~200 μm of each other. Each pipette was backfilled with mineral oil and connected to a 5 μL glass syringe as above. One pipette contained ACSF with APV and 10 μM Alexa-594, and the other contained ACSF with APV, ZX1, and 50 μM Alexa-594. We inserted the tips of both pipettes into the small craniotomy as above. We measured control sound-evoked responses, infused APV and remeasured these responses, and then infused APV and ZX1 and measured these responses a third time. We monitored the diffusion of each solution in the cortex by the increase in red transcranial fluorescence as above. We followed a similar approach for the sequential infusion of vehicle and ZX1 in We performed transcranial imaging to locate A1 in each mouse. We removed the isoflurane from the oxygen flowing to the animal and began imaging sound-evoked responses at least ten minutes later . Sounds ΔF) to the baseline fluorescence (F), where F is the average fluorescence of 1 s preceding the sound onset (for each pixel in the movie). We applied a two-dimensional, low-pass Butterworth filter to each frame of the ΔF/F movie, and then created an image consisting of a temporal average of 10 consecutive frames (0.5 s); the temporal average started at the end of the sound stimulus. This image indicated two sound-responsive regions corresponding to the low frequency tonotopic areas of A1 and the AAF (ΔF to the F of the ROI to yield ΔF/F responses. ΔF/F responses from 3 to 5 presentations of the same sound level and frequency were averaged. Response amplitude was the peak (50 msec window) of the transcranial response that occurred within one second of the sound onset. To quantify of the effects of ZX1 on the type of gain change (multiplicative/divisive and additive/subtractive) the response amplitudes from each mouse (in control and ZX1) were normalized to the largest response in control. The normalized response to each sound in ZX1 was then plotted against the corresponding control response, and the slope and y-intercept were quantified with a regression line fit through the data.To localize A1, we used 50 or 60 dB SPL, 5 or 6 kHz tones and we normalized the sound-evoked change in fluorescence after sound presentation ( the AAF . A regio2) over A1 for improved optical access, and inserted the pipette containing ZX1 into the cortex at the edge of this craniotomy. Mode-locked infrared laser light  was delivered through a galvanometer-based scanning 2-photon microscope  controlled with scanimage  following zinc chelation by plotting the sound-evoked response amplitudes in ZX1 against the corresponding response amplitudes in control conditions. We quantified the slope and y-intercept of this relationship for each neuron with a major axis regression . The slices were then transferred and incubated at 36°C in ACSF (see above) for 30 min. Subsequently, slices were transferred to a solution containing 4% paraformaldehyde  in 0.01M phosphate buffered saline (PBS) and incubated at 4°C overnight. Following this fixation, slices were rinsed five times in PBS and mounted on glass slides. We acquired fluorescent images of GCaMP6f expressing neurons in cortical slices with 2-photon microscopy (see above).To image GCaMP6f expressing neurons in brain slices, mice that had undergone AAV injections were deeply anesthetized with isoflurane and decapitated. Brains were quickly removed and sectioned, with a vibratome , into 300 μm acute slices containing the temporal cortex. We prepared the slices in a solution containing (in mM): 2.5 KCl, 1.25 NaHExperiments with WT and ZnT3 KO were blinded.Analysis was performed with MATLAB  and QuickCalcs . Group data are presented as mean ± standard error of the mean. Pairwise comparisons between groups were performed with the Student’s paired t-test, t-test or one sample t-test  or the Wilcoxon signed-rank or rank sum tests . Normality of the distribution of data was assessed with the Lilliefors test. Significance is defined as p<0.05. The sample size for experiments was chosen to be consistent with similar studies in the field, such as in . 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 reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.Overall, the reviewers felt that this is an intriguing study and were convinced that you had shown an effect of zinc chelation on auditory processing. They were also impressed by the range of approaches used and stated that these findings are likely to inspire follow up work in this field. The reviewers did, however, raise a large number of issues, with several requests for additional data. Reviewer #2 highlighted the lack of mechanistic insights provided by the current data, while reviewer #3 was concerned that the effects of zinc removal are restricted to loud sounds and that the data are too variable to make firm conclusions about gain control. Both reviewers pointed out that it is not clear that (or at least to what extent) the effects described necessarily indicate an involvement of synaptically released zinc. While recognizing the amount of work that has already gone into this study and that it is obviously not possible to cover everything in one article, it was clear from the subsequent discussion between the reviewers that the study currently contains many loose ends and that these could not easily be fixed in a revision. Unfortunately, we have therefore concluded that we will not be able to consider your paper further at Reviewer #2:This paper shows interesting effects of zinc chelation – or removal of synaptically released zinc via genetic deletion of the vesicular zinc transporter ZnT3 – on auditory processing in mice. Disrupting zinc signaling is shown to have effects at different levels: 1) apparently (see below) knocking out ZnT3; 2) Responses to loud (>70 dB) sound stimuli in A1 neuronal populations is increased by ZX1 or ZnT3 deletion.These results show quite clearly that zinc modulates elements involved in auditory processing. A significant strength of this paper is that effects are shown in several different experimental paradigms that examine auditory signaling at several levels. Clearly, this laboratory is poised to examine how synaptic mechanisms influence auditory processing in physiologically and behaviorally relevant ways. A significant weakness of the paper is that little insight is provided into the actual synaptic mechanisms within the auditory pathway that underlie the observed effects of zinc. The authors claim at the beginning of the Discussion that their "results provide fundamental synaptic mechanisms governing normal neural processing." I think this is a significant overstatement. The results don't even show directly that the primary effects of zinc are actually at synapses . If we assume that the primary effects occur at excitatory synapses, we don't know whether these synapses are on principal neurons, interneurons, or both, and whether the effects reflect primarily modulation of AMPA or NMDA receptors.The authors recognize these gaps and make the reasonable point that further experiments are required to elucidate the underlying mechanisms (Discussion paragraph six). I think that the experiments presented here provide ample motivation for those future experiments, and I accept the implication that the more mechanistically detailed experiments are beyond the scope of the present paper. I would, however, ask the authors to tone down the language regarding insights into fundamental mechanisms.Reviewer #3:General Comments: I think the biggest problem I have with the results is that it is very puzzling that the effects of synaptic zinc are restricted to loud sounds > 70 dB – And the authors seem to try to explain this odd and selective effect to loud sounds with a lot of cursory commentary. Finally I feel that the authors extrapolate too much from these limited results to autism and schizophrenia in their Discussion.A bit more detail:1) 2) The authors show that effects for 70 and 80 dB in 3) All results in normal mice are based on inference from the effects of ZX1 – which chelates and thus reduces synaptically evoked zinc, but presumably also reduces extracellular Zn as well. The authors should provide clearer evidence of the relative effects on synaptically evoked Zn and extracellular Zn level with careful measurements. It would also be helpful to have a more direct technique to specifically enhance the concentration of Zn in synaptic transmitter vesicles. Would additional synaptic evoked zinc release lead to even more inhibition of A1 responses to loud sounds? Or to effects on a broader range of intensities? Are there mouse mutants with enhanced Zn concentration in synaptic vesicles?4) What about layer II, III cells with non-monotonic intensity tuning? Do they also show the effects of ZXI as cells with monotonic intensity tuning?5) The authors focused on layer II, III cells – what about layer IV cells? Are the effects of Zn at loud intensities present only for the supragranular cells or also observed in layer IV cells, which presumably are activated primarily from the thalamic inputs?6) In general, one wonders how important ethologically these Zn effects would be for mice – given that most sounds in their normal acoustic environment are less than 70 dB.7) What is the clinical evidence that schizophrenics are selectively impaired in sound perception at loud intensities of sound? My impression is that schizophrenics are often hypersensitive to all sounds, including faint ones.Obviously a lot of work went into this study. The findings are intriguing, but the story feels incomplete.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Andrew King as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Richard Dyck (Reviewer #2).Thank you for submitting your article "Cell-specific gain modulation by synaptically released zinc in cortical circuits of audition" 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:The reviewers agreed that this study makes an important and substantial contribution to elucidating the possible role of synaptically released zinc in the normal function of auditory cortex, and that it is likely to inspire future studies in this area. Most of the existing literature has focused on the involvement of synaptic zinc in relation to pathological conditions. By showing that synaptic zinc can change the gain of sound-evoked responses in a neuron-specific fashion, this work provides clear evidence for a contribution to normal brain function. It is therefore likely to have a significant impact on the field of zinc neurobiology as a whole.Essential revisions:The reviewers agreed that the manuscript is, generally, clearly written and logically organized, and substantially improved relative to the previous version. The following issues were raised:1) There was some discussion among the reviewers about the selectivity and specificity of ZX1 binding. While it was agreed, on the basis of previous work, that the effects of ZX1 are unlikely to be caused by chelating extracellular magnesium or calcium, this issue should be addressed in the manuscript, particularly as two-photon calcium imaging was used to measure the activity of cortical neurons.2) The authors assume that, at high sound intensities, zinc inhibition of PV interneurons leads to potentiation of the principal neurons, and this effect is NMDA-dependent, because this relationship is not seen when an NMDA antagonist is applied. Please comment on the NMDA-dependent mechanism by which the PV interneurons are normally inhibiting the principal neurons.3) The bar graphs showing average change in fluorescence per dB should be removed, since the information they provide is either redundant or confusing. For example, the bar graph in 4) The authors should make it clear that their data are specific to circuits in layer 2/3 of auditory cortex. Considering the differential innervation of zincergic inputs in different layers, together with the differential distribution of PV , SOM  and VIP  interneurons in auditory cortex, as well as unique within-column and inter-columnar information processing circuits, their results may not apply to the deeper layers of the cortex. [Editors’ note: the author responses to the first round of peer review follow.]Overall, the reviewers felt that this is an intriguing study and were convinced that you had shown an effect of zinc chelation on auditory processing. They were also impressed by the range of approaches used and stated that these findings are likely to inspire follow up work in this field. The reviewers did, however, raise a large number of issues, with several requests for additional data. Reviewer #2 highlighted the lack of mechanistic insights provided by the current data, while reviewer #3 was concerned that the effects of zinc removal are restricted to loud sounds and that the data are too variable to make firm conclusions about gain control. Both reviewers pointed out that it is not clear that (or at least to what extent) the effects described necessarily indicate an involvement of synaptically released zinc. While recognizing the amount of work that has already gone into this study and that it is obviously not possible to cover everything in one article, it was clear from the subsequent discussion between the reviewers that the study currently contains many loose ends and that these could not easily be fixed in a revision. Unfortunately, we have therefore concluded that we will not be able to consider your paper further at eLife.1) To address the lack of mechanistic insights we collected and analyzed new data to uncover the cellular and molecular mechanisms underlying zinc-mediated gain modulation in the auditory cortex. Namely, we performed new experiments to investigate the effect of synaptic zinc on the gain of principal neurons, as well as interneurons  Regarding the issue that the effects of zinc removal are restricted to loud sounds, our new experiments and analyses now show effects of zinc at low (30 dB SPL) and high sound intensities  Regarding the issue of providing firm data for gain control, 4) To address the issue of the extent of the involvement of the synaptically released zinc, we performed additional analysis of our results, as suggested by reviewer 2. To track the origin of the extracellular zinc modulating A1 gain control, we studied A1 gain control in ZnT3 KO mice, which lack the vesicular zinc transporter ZnT3 and synaptic zinc . Compared to WT mice, ZnT3 KO mice showed increased change of the neuronal response per 10 dB SPL , suggestAdditionally, to track the origin of the extracellular zinc increasing the gain of principal neurons, we performed new experiments to study the effects of ZX1 in ZnT3 KO mice, which had been injected with AAV-GCaMP6fCaMKII. Zinc chelation had no effect on the sound-evoked responses of principal neurons in ZnT3 KO mice, indicating that synaptic zinc increases the gain of principal neurons .Based on the editorial and reviewer’s comments, we opted to focus our manuscript on the role of synaptic zinc on the gain control of A1 principal neurons and interneurons. Our results highlight synaptic zinc as a novel modulator of gain control in the auditory cortex, and provide insights on the cellular and molecular mechanisms underlying this modulation.The effects of zinc on the frequency tuning of A1 principal neurons and interneurons; as well as the effects of zinc on frequency discrimination will be the focus of our next manuscript.Reviewer #2:[…] These results show quite clearly that zinc modulates elements involved in auditory processing. A significant strength of this paper is that effects are shown in several different experimental paradigms that examine auditory signaling at several levels. Clearly, this laboratory is poised to examine how synaptic mechanisms influence auditory processing in physiologically and behaviorally relevant ways. A significant weakness of the paper is that little insight is provided into the actual synaptic mechanisms within the auditory pathway that underlie the observed effects of zinc. The authors claim at the beginning of the Discussion that their "results provide fundamental synaptic mechanisms governing normal neural processing." I think this is a significant overstatement. The results don't even show directly that the primary effects of zinc are actually at synapses . If we assume that the primary effects occur at excitatory synapses, we don't know whether these synapses are on principal neurons, interneurons, or both, and whether the effects reflect primarily modulation of AMPA or NMDA receptors.1) To address the lack of mechanistic insights, we performed new experiments and analyses that provide insight into the cellular and molecular mechanisms underlying the observed effects of zinc –5. For m2) We agree with the reviewer’s comment on the “overstatements”, and we modified the Discussion accordingly. Moreover, we removed the overstatements throughout the manuscript. Given the new mechanistic experiments, we now know that the zinc-mediated modulatory effects occur at principal neurons and interneurons; and non-NMDA receptors, as well as NMDA receptors, are involved in this modulationThe authors recognize these gaps and make the reasonable point that further experiments are required to elucidate the underlying mechanisms (Discussion paragraph six). I think that the experiments presented here provide ample motivation for those future experiments, and I accept the implication that the more mechanistically detailed experiments are beyond the scope of the present paper. I would, however, ask the authors to tone down the language regarding insights into fundamental mechanisms.We agree with the reviewer and we removed and/or toned down the language throughout the manuscript, regarding into fundamental mechanisms.Reviewer #3:I think the biggest problem I have with the results is that it is very puzzling that the effects of synaptic zinc are restricted to loud sounds > 70 dB – And the authors seem to try to explain this odd and selective effect to loud sounds with a lot of cursory commentary. Finally I feel that the authors extrapolate too much from these limited results to autism and schizophrenia in their Discussion.1) Our new experiments and analyses –5 now sh2) We agree with the reviewer about autism and schizophrenia and we removed these topics in the revised Discussion.1) See point-by-point response to the Editors’ comments, response 32) The authors show that effects for 70 and 80 dB in Due to the focus of our revised manuscript on the role of synaptic zinc on the gain control of A1 principal neurons and interneurons, we removed 3) All results in normal mice are based on inference from the effects of ZX1 – which chelates and thus reduces synaptically evoked zinc, but presumably also reduces extracellular Zn as well. The authors should provide clearer evidence of the relative effects on synaptically evoked Zn and extracellular Zn level with careful measurements.See point-by-point response to the Editors’ comments, response 4.It would also be helpful to have a more direct technique to specifically enhance the concentration of Zn in synaptic transmitter vesicles. Would additional synaptic evoked zinc release lead to even more inhibition of A1 responses to loud sounds? Or to effects on a broader range of intensities? Are there mouse mutants with enhanced Zn concentration in synaptic vesicles?This is an important point and we are very interested in investigating how increased levels of synaptic zinc affect the sound-evoked responses of A1 neurons. Experiments addressing this issue are outside the scope of this manuscript and will require new tools, but will be pursued in future studies in our lab.4) What about layer II, III cells with non-monotonic intensity tuning? Do they also show the effects of ZXI as cells with monotonic intensity tuning?This comment was not very clear to us. Based on our understanding, we assume that the reviewer asks what is the effect of ZX1 on the frequency tuning of layer II, III cells with monotonic and non-monotonic frequency tuning. Due to the focus of our revised manuscript on the role of synaptic zinc on the gain control of A1 principal neurons and interneurons, we removed all the graphs from the manuscript that were focused on frequency tuning.5) The authors focused on layer II, III cells – what about layer IV cells? Are the effects of Zn at loud intensities present only for the supragranular cells or also observed in layer IV cells, which presumably are activated primarily from the thalamic inputs?In the revised manuscript we added new experiments and analyses showing that the effects of synaptic zinc on cortical gain are cell-type specific within layer 2/3. We are currently investigating the effects of zinc on different cell-types in L4 and L5. These experiments are outside the scope of this manuscript, but will be pursued in future studies in our lab.6) In general, one wonders how important ethologically these Zn effects would be for mice – given that most sounds in their normal acoustic environment are less than 70 dB.In our new experiments and analyses addressing the effect of zinc on the sound-evoked responses of different cell types , 4 and 57) What is the clinical evidence that schizophrenics are selectively impaired in sound perception at loud intensities of sound? My impression is that schizophrenics are often hypersensitive to all sounds, including faint ones.Due to the focus of our revised manuscript on the role of synaptic zinc on the gain control of A1 principal neurons and interneurons, we removed from our discussion the topics of schizophrenia and autism.Obviously a lot of work went into this study. The findings are intriguing, but the story feels incomplete.We performed many additional experiments and analyses; we included several new figures and analyses –5 and ed[Editors' note: the author responses to the re-review follow.]1) There was some discussion among the reviewers about the selectivity and specificity of ZX1 binding. While it was agreed, on the basis of previous work, that the effects of ZX1 are unlikely to be caused by chelating extracellular magnesium or calcium, this issue should be addressed in the manuscript, particularly as two-photon calcium imaging was used to measure the activity of cortical neurons.To address this concern, we included this paragraph in the revised manuscript (Results section):“Consistent with the high selectivity of ZX1 over calcium and other biologically relevant metal ions such as magnesium , the lack of ZX1 effects on the sound-evoked responses in ZnT3 KO mice further validate that the observed effects of ZX1 on the sound evoked responses of WT mice are due to the chelation of synaptic zinc – and not to the potential non-specific effects of ZX1 on calcium or other metal ions.”2) The authors assume that, at high sound intensities, zinc inhibition of PV interneurons leads to potentiation of the principal neurons, and this effect is NMDA-dependent, because this relationship is not seen when an NMDA antagonist is applied. Please comment on the NMDA-dependent mechanism by which the PV interneurons are normally inhibiting the principal neurons.To address this concern, we included this paragraph in the revised manuscript (Discussion section):“Blockade of NMDARs with APV reduced the sound-evoked responses of PV neurons  and incr3) The bar graphs showing average change in fluorescence per dB should be removed, since the information they provide is either redundant or confusing. For example, the bar graph in To address these concerns, in our revised figures we removed these bar graphs from 4) The authors should make it clear that their data are specific to circuits in layer 2/3 of auditory cortex. Considering the differential innervation of zincergic inputs in different layers, together with the differential distribution of PV , SOM  and VIP  interneurons in auditory cortex, as well as unique within-column and inter-columnar information processing circuits, their results may not apply to the deeper layers of the cortex.To address this concern, we included this paragraph in the revised manuscript(Discussion section):“Our findings are mostly specific to cells and circuits in L2/3. […] Future studies will be required to address the effects of synaptic zinc on the gain of neurons and circuits located outside of L2/3.”


With the increasing interest in the potential benefits of nanotechnologies, concern is still growing that they may present emerging risks for workers. Various strategies have been developed to assess the exposure to nano-objects and their agglomerates and aggregates (NOAA) in the workplace, integrating different aerosol measurement instruments and taking into account multiple parameters that may influence NOAA toxicity. The present study proposes a multi-metric approach for measuring and sampling NOAA in the workplace, applied to three case studies in laboratories each dedicated to materials with different shapes and dimensionalities: graphene, nanowires, and nanoparticles. The study is part of a larger project with the aim of improving risk management tools in nanomaterials research laboratories. The harmonized methodology proposed by the Organization for Economic Cooperation and Development (OECD) has been applied, including information gathering about materials and processes, measurements with easy-to-use and hand-held real-time devices, air sampling with personal samplers, and off-line analysis using scanning electron microscopy. Significant values beyond which an emission can be attributed to the NOAA production process were identified by comparison of the particle number concentration (PNC) time series and the corresponding background levels in the three laboratories. We explored the relations between background PNC and microclimatic parameters. Morphological and elemental analysis of sampled filters was done to identify possible emission sources of NOAA during the production processes: rare particles, spherical, with average diameter similar to the produced NOAA were identified in the nanoparticles laboratory, so further investigation is recommended to confirm the potential for worker exposure. In conclusion, the information obtained should provide a valuable basis for improving risk management strategies in the laboratory at work. In the last decade nanotechnologies (NTs) and nanomaterials (NMs) have seen rapid development worldwide, as seen in the growing numbers of R&D investments , nanotecNMs are defined by the International Organization for Standardization (ISO)  as materAfter 10 years of studies and research in this field, Maynard and Aitken  affirm tThe toxicological literature suggests that multiple parameters may influence NMs health effects: their size, shape, particle number and mass concentration, surface area, aggregation and agglomeration, water solubility, and surface chemistry are the most important metrics in exposure studies ,9,10.Different strategies have been developed to assess airborne NMs and NOAA in the workplace, based on the integration of different aerosol measurement instruments providing NOAA exposure information in occupational settings ,12,13. M(1)Far-Field (FF) approach: background is measured in a place not influenced by the process, in the same facility, but far from the workplace where NMs are produced. Some authors also consider the FF background measurements as a “spatial approach”, in which the difference between the background and workplace concentrations can be attributed to the work with the NOAA investigated. FF background measurements should be collected simultaneously with the NOAA measurements .(2)Near-Field (NF) approach: based on monitoring before work, at the same location as the nano-workstation. The NF background is also defined as a “time-series” approach, assuming that the concentration determined when there is no ongoing work is the background concentration and any increases during work can be attributed to the process.The distinction of background from NOAA emission specifically related to the process plays a key role in any analysis of workers’ exposure. Two main approaches for background characterization are frequently used in measurement studies :(1)Far-Further studies include measurements of outdoor background to better characterize the exposure scenario .Therefore, international research efforts are aimed at defining standard methods for risk characterization and exposure assessment ,17. On tThe OECD document  suggesteThe main objective of the present study was to propose a multi-metric approach for NOAA measurement and sampling, based on the harmonized and tiered OECD methodology; it was applied in three NOAA case studies with different shapes and dimensionality: graphene (G), a typical two-dimensional (2D) material, nanowires (NW) having a one-dimensional (1D) structure, and nanoparticles (NP) considered as having zero dimensionality (0D). The case studies were conducted at the NEST Laboratories in Pisa, Italy; these research facilities are very active in responsible development of nanotechnologies and have also conducted further studies to investigate the potential exposure of their personnel involved with NOAA .www.nano-lab.it), in which field measurements will be employed to integrate and improve CB risk management for workers in the research laboratories involved with NOAA. Although all three OECD tiers have been conducted in line with the final aim of the project, the present study focuses on the results of the first and second tiers in order to assess the effectiveness of their application to the case studies.The present study is part of a larger research project called “Nano-Lab” (2) core shell nanoparticles containing an aurum (Au) nanostructure (NP). The NOAA physical–chemical characteristics such as shape, size, composition, production method and others, are reported in the technical data sheets  equipped with an energy dispersive spectroscopy system (EDS) to collect morphological and chemical information on NOAA trial materials. Two SEM images of G are reported in SEM images in 2 core shells. The bright dots are generated by Au nanostructures embedded in the shell, even though the EDS analysis gave a low Au signal  with a hand-held condensation particle counter (CPC) and air sampling using a Sioutas personal impactor whose filters were analyzed off-line by SEM. Particular attention was directed toward any outlier in the time series measured in the workplace, in relation to the background levels. In these cases, to make the data more reliable, we compared PNC measurements carried out simultaneously by CPC and a fast mobility particle sizer (FMPS). Indoor climatic parameters were continuously monitored during all measurements and sampling. Further information about the measurement strategy and instrument features is reported in the Materials and Methods section.-3, was much closer to the lower edge whereas on day 6 the median, at 1029 #/cm3, was closer to the upper edge of the box;on day 5 the median, at 907 #/cm-3 and on day 6 it was 164 #/cm3) were not dissimilar.the distribution dispersions (interquartile distance on day 5 was 123 #/cmBox plots of FF background show an asymmetric distribution of PNC:outdoor) box plot shows a symmetric distribution of PNC with the highest values (median 6420 #/cm3 and interquartile distance 1858 #/cm3). The NF background (bkgNF) box plots too show symmetric distribution; medians are 2829, 2904 and 1997 #/cm3 and the corresponding interquartile distances are 196, 493 and 106 for the NW, G and NP laboratories.The outdoor background  are summarized in 3, with the corresponding standard deviation of 167 #/cm3 related to two days of measurements, was considered as FF background (bkgFFavg).Mean PNC and standard deviation (σ bkg) of PNC measured by CPC in the FF, outdoor background air and in the NF of the G, NW and NP laboratories before the processes  bkg values of PNC are very similar, the corresponding variances are different; furthermore, in the G laboratory the NF σ bkg value is higher than the others. This last variability can be attributed to research staff passing through frequently for work-related activities in the laboratory and therefore near the CPC sampling point; this was observed in both the G and NW laboratories during the background data collection interval. In the NP laboratory the σ bkgoutdoor are the highest recorded values, essentially influenced by environmental pollution, vehicles and motorcycles passing near the CPC collection point during the measurements.Last, mean and σ bkgTime series of workplace PNC, measured on one day in each case study, summarizing all the process phases, are reported in In the G laboratory a, measurIn the NW laboratory b, the meLOD 0.2–0.4 wt %) so we have no evidence of the Au nanostructures inside the SiO2 shell.SEM images of the filters sampled by Sioutas worn by workers during each production process did not detect any materials attributable to those produced in the G and NW laboratories. The morphological investigations of D (250–500 nm) and backup (<250 nm) sampled filters in the NP laboratory revealed rare particles which, on account of their spherical shape and average diameter, can be considered similar to the NP produced. EDS analysis of these spherical particles  reactor opening) and 3.1 (CVD reactor cleaning) of the process in the G laboratory show no peak intensity attributable to G production. Moreover, the process phase 1 shows decreases in PNC that might be related to a pure nitrogen flux originating from the CVD reactor during the vacuum chamber opening phase, which tends to lower the particulate concentration in the area of the laboratory where air is sampled. For process phases 2 (CVD growth) and 4 (cleaning the CVD reactor components in the furnace), it is interesting that the variations in the PNC are associated with events unrelated to the G production; as also reported in the workbook, these events can be traced back to the laboratory door being opened or closed, or related to staff movements and/or room cleaning.The process phase 3.2, related to graphite spraying on some reactor components, shows a peak whose intensity is well above the significant value of PNC in the G laboratory; this peak was not associated with the G production process but was directly related to the graphite spraying. In addition, the off-line SEM investigations do not indicate any G particles on the filters collected by the Sioutas.In the NW laboratory the CPC real-time measurements showed no changes in PNC associated with phase 3 (sample cleavage in the glove box). It was also evident that in the process phase 1  growth) there were features whose intensity was well above the significant value established for this laboratory; this high intensity might be associated with events not related to NW production, as mentioned for the G case study, because the NW growth occurs in a closed reactor under vacuum. Otherwise, during phase 2 (sample loading/unloading in the CBE load lock and sample mounting/unmounting in the glove box) there was a clear increase of the concentrations in the operator’s personal breathing zone (PBZ)—as defined by the European Committee for Standardization (CEN) —althoughIn the NP laboratory during a small part of the process phases 1 (synthesis in liquid) and 4 (drying) the PNC signal exceeded the significant value for this laboratory. Even sub-phase 4.1 (vacuum pump turned on/off), in which there was a fast change in PNC c, inset,In this case, the background contribution heavily influenced the PNC as measured by CPC in the NP laboratory. Compared to the previous cases, a different type of analysis can be proposed to take account of the background contribution: this would be to extrapolate the background contribution. In addition we compared CPC time series to microclimatic parameters measured by the BABUC-A probes, with particular reference to the relative humidity (RH) throughout the sampling period. After about 60 min from starting the measurement, the background contribution curve was strongly correlated with the RH curve, as clearly shown in The “residual” curve in 2 shells; however, the EDS probe did not detect the Au signal and there was no evidence of Au nanostructures in the FE-SEM image, probably because of the small amount of metal in each core shell. EDS microanalysis is a powerful and useful form of elemental analysis; however, it suffers some limitations: the limit of detection is generally above 1000–3000 ppm, but can vary for different elements, matrixes and analytical lines, and the spatial resolution is usually above 1 μm3 [w/w) is too small to be detected and spatially resolved by the EDS micro-probe. However, since the EDS Au signal was also weakly detected in NP trial samples  risk management are needed. Semiconductor 1D nanowires may also raise critical issues for OSH on account of their threadlike shape and similarities with hazard questions related to fibers. Silica 0D NP are widely used and already included in a range of nano-enabled consumer products, with an increasing impact on exposed populations.The different production processes conducted in the three R&D laboratories  at the s−1 mbar before starting a process.Sample preparation and loading. The reactor chamber is vented and the reactor lid is lifted manually; the sample (up to 10 × 10 mm) is placed on the graphite heater inside the reactor; the chamber is closed and pumped up to 5 × 102.1.Hydrogen etching. SiC substrates are treated with hydrogen etching at a temperature of around 1200 °C and a pressure of 450 mbar for a few minutes, in order to remove polishing scratches and obtain atomically flat terraces.2.2.Thermal decomposition. The hydrogen etched substrates are heated in an argon atmosphere at a temperature above 1300 °C and a pressure of 780 mbar for 10–15 min.CVD Growth. The growth process can be divided into two steps, both conducted in a commercial resistively heated cold-wall reactor (Aixtron HT-BM):Reactor cleaning. The quartz and ceramic parts are periodically cleaned in an oven operated in air, in order to remove any carbon deposit. During the cleaning some reactor components are restored by graphite spraying.Cleaning in the furnace. The parts are heated at 950 °C for at least one hour.The process can be summarized in the following phases.2 (about 120 m3 volume), with a mechanical ventilation system producing an air change of 3–6 volumes per hour. Phases 2 and 4 are run in a closed system. The process involves two or three workers equipped with personal protective devices .Each cycle produces a few tens of micrograms of G . The G laboratory has an area of 40 m−10 Torr.CBE growth. NW are synthesized through epitaxial growth techniques, e.g., the CBE, on a macroscopic crystalline substrate made of a semiconductor material, Si or InAs . Usually2.1.−8 Torr can be achieved in 1–2 h. The cassette is then transferred into the preparation chamber, and after that to the growth chamber, for NW synthesis.Sample mounting and loading (before CBE growth). The substrate is first cleaved into small pieces of about 1 cm × 1 cm, and then fixed on a sample holder made of Mo through In-bonding inside a glove box. After that, the sample holder is placed in a cassette and transferred into the CBE system via a load-lock. The load-lock is pumped by a turbo-molecular pump and a base pressure of 102.2.Sample unloading and unmounting (after the CBE growth). The plates with the grown samples are transferred from the growth chamber into the preparation chamber, mounted on the cassette that is transferred into the load-lock, and then again into the glove box, where the sample holder is placed on a hot plate at about 350 °C, to allow the In to melt, and the sample can be removed from the Mo plate. Frequently, a new sample mounting and loading (phase 2.1) is done immediately after the sample removal (phase 2.2).Sample loading. This phase is divided into two following steps:Sample cleavage. The next phase is cleavage of the sample, for its morphological characterization by SEM.NW production can be summarized in the following phases:The CBE chamber needs periodical maintenance, to clean the different parts of the system and inside the reactor. This is done by highly qualified staff, at least once a year.2 (about 60 m3 volume), with a mechanical ventilation system producing an air change of 3 volumes per hour; an automatic system for aspiration/cleaning starts in case of emergency. Phase 1 is in a closed system (ultra-vacuum chamber); phases 2–3 are carried out in a ventilated glove box. Two or three workers equipped with personal protective devices  are involved in the process.Each cycle produces about 20 μg of NW with an estimated total laboratory production of 15 mg/year. The NW laboratory has an area of 20 mSynthesis. The nano-architectures are synthesized by a wet chemical approach. A yellow solution of chloroauric acid underwent fast reduction by sodium borohydride in the presence of poly(sodium 4-styrene sulfonate) (PSS), with vigorous stirring, resulting in a deep orange colloidal solution of negatively charged gold NP, less than 3 nm in diameter.l-lysine) (PL).Aggregation. The 3 nm gold NP are then assembled in spherical arrays by controlled aggregation achieved by ionic interaction with positive poly.Four different phases of the NP production process are:2 (about 90 m3 volume), with a mechanical ventilation system producing an air change of 3 volumes per hour. Phases 1 and 4 are carried out in a chemical ventilated hood. One worker equipped with personal protective devices  is involved in the process.The NP laboratory has an area of 30 mA measurement strategy based on a harmonized tiered approach  has been3) of particles from 10 nm to 1 µm, with 1 s time resolution and accuracy ±20%; .Condensation particle counter  to measure in real-time the number concentration (#/cm3) of particles from 250 nm to 2.5 µm (five stages).Personal samplers  equipped with a pump  for concentrations (#/cmField emission scanning electron microscope (FE-SEM) Ultra Plus (ZEISS) with EDS probe Inca 250-X-Max50 and INCA mapping software in line scan mode. The microscope is equipped with a modified Gemini column and has four different detectors: in-chamber Everhat–Thornely SE for surface topography, in-column In lens best for high efficiency, angle-selective backscattered detector ASB for material contrast and topographical information, in-column ESB for material contrast even at low KV.Microclimatic probes integrated in a control unit  to measure in real time physical parameters such as temperature, RH and air speed.The second tier included workplace walkthrough to obtain information about facilities, equipment, natural and mechanical ventilation and air change systems, production processes and phases, working times, collective and personal protective devices. Measurements were taken and air sampled. The instruments included easy-to-use and hand-held real-time devices, personal samplers and electronic microscopy for off-line analysis:Fast mobility particle sizer  to characterize real-time size distribution and simultaneously measure total particle numbers and mass concentration, in the size interval 5.6–560 nm, with 1 s time resolution;2/cm3) of particles from 10 nm to 1 µm, with 1 s time resolution, corresponding to the tracheobronchial (TB) or alveolar (A) pulmonary fractions, based on the model published by the International Commission on Radiological Protection [Nanoparticle surface area monitor  to measure average and cumulative surface area  to measure polycyclic aromatic hydrocarbons (PAHs) surface-adsorbed on carbon aerosol with aerodynamic diameter from 10 nm to 1.5 µm, with a response time of 10 s in a measuring interval from 0 to 1000 ng/mOzone analyzer  to measure ozone levels in the air.Nano micro orefice uniform deposit impactor , equipped with a rotary pump , to collect particles in a dimensional range from 10 nm to 10 µm.Inductively coupled plasma mass spectrometry .Inductively coupled plasma optical emission spectroscopy .Atomic fluorescence spectrometer .The third tier consisted of an extensive measurement campaign with real-time instruments and time-integrated samplers . The samples were then fully analyzed off-line. The instruments included the same set-up as for Tier 2 with the following added devices:An intensive measurement campaign was conducted, including all real-time and time- integrated instruments listed . Workers wore the personal samplers during their activities .Measurements and sampling were done on two days for each case study: day 1–2 NW a, day 3–A room with the same volume, orientation, and structural and ventilation properties as the laboratories was selected for the indoor FF background characterization d: FF bacTime-sheets of measurements in chronological order associated with each single phase of the processes and any further external event that arose during the activities were all entered in the daily register (study workbook).We studied three exposure scenarios of production of NOAA with different dimensionalities—2D G, NW with a 1D structure and NP with a 0D shape, applying tiers 1 and 2 of the OECD harmonized methodology , to asseFF values were compared to bkgNF measured before starting the NOAA production in the three laboratories. Since the bgkFF was always lower than the bkgNF in all three laboratories, we took the bkgNF values plus three times the corresponding standard deviations as significant, above which the CPC measurement can be attributed to the NOAA production process.We also looked closely into the background characterization in order to identify the significant PNC values for each laboratory. The FF background was measured in a room with structural characteristics similar to the study laboratories, during two non-working days. We adopted this approach as an alternative to simultaneous measurement of FF background and NOAA in order to overcome limitations related to the availability of instruments; bkgNF values were different in the three laboratories and this variability was probably influenced by the frequent access of research staff for work-related activities in the laboratory and near the CPC sampling point during the background data collection interval; this particular aspect may need further specific analysis.The σ bkgIn the NP laboratory we examined a different approach to assess the background contribution because the PNC measured by CPC was closely correlated to the RH. In this respect, it will be interesting to clarify the relations between background PNC and microclimatic parameters, as a recommendation for further research.In general, we concluded that to completely characterize the exposure scenarios of NOAA production in the three case studies, additional resources in terms of instrumentation and a more detailed analysis (as envisaged by the OECD in tier 3) will be required; this might imply considering the size distribution, the surface areas of the particulate matter, the concentrations of the PAHs and other pollutants and, finally, chemical and morphological analysis using TEM on ad hoc collected samples.-G laboratory. Real-time measurements showed high PNC associated with phase 3.2 of graphite spraying during the CVD reactor cleaning, but SEM did not detect any produced G on the sampled filters. As a precautionary measure, this specific phase should be conducted under an aspiration hood or in a glove box.-NW laboratory. Although the CPC measurements showed a rise in PNC during phase 2 (sample mounting and loading/unloading and unmounting), SEM did not detect any NW on the Sioutas filters. Although the PNC was probably related to sublimation of In from the hot Mo plate, an ad hoc investigation is called for to properly analyze the chemical and physical parameters of the emission source in this specific phase. In any case, the measurements made inside the glove box demonstrated the effectiveness of this containment measure. The study of the CBE reactor cleaning phase (not included in the present measurement campaign), might be useful to analyze the whole process so as to finally exclude workers’ exposure to NW.-2 particles (with an approximate diameter of 100 nm) similar to the hollow silica shells produced but with no evidence of the Au nanostructures inside. This implies that further investigation is needed to confirm whether such NPs are in fact released by the process and in which specific phase this release might occur.NP laboratory. In a large part of the process, PNCs were greater than the significant value identified for this laboratory. In this case the background contribution heavily influenced the PNC, as measured by CPC, so we proposed a different type of analysis to take account of the background. By comparison of CPC time series with microclimatic parameters measured with BABUC-A probes, with particular reference to the RH throughout the sampling period, we found a structure corresponding to phase 4.1 (drying vacuum pump turned on) that exceeded the background curve; the PNC increase in this case is probably related to the pump switching and not strictly connected to NP production. However, SEM of sampled filters showed there were rare spherical SiOSpecific findings for each case study are reported as follows:In conclusion, also according to the findings of the complementary study on workers’ personal exposure conducted in the same research facilities  and to tThe measurement strategy may be used to study workers’ exposure to NOAA also in industrial production processes, with promising applications in the near future. Its integration in a prevention-through-design model, taking into account the cost and effectiveness of workplace monitoring techniques, will contribute to the responsible development and use of nano-products.