Weight loss recovers MOR function, but it remains unknown whether it also recovers aberrant opioid-dopamine interaction. Here we addressed this issue by studying 20 healthy non-obese and 25 morbidly obese women (mean BMI 41) eligible for bariatric surgery. Brain MOR and D2R availability were measured using positron emission tomography (PET) with [11C]carfentanil and [11C]raclopride, respectively. Either Roux-en-Y gastric bypass or sleeve gastrectomy was performed on obese subjects according to standard clinical treatment. 21 obese subjects participated in the postoperative PET scanning six months after bariatric surgery. In the control subjects, MOR and D2R availabilities were associated in the ventral striatum (r = .62) and dorsal caudate (r = .61). Preoperatively, the obese subjects had disrupted association in the ventral striatum (r = .12) but the unaltered association in dorsal caudate (r = .43). The association between MOR and D2R availabilities in the ventral striatum was recovered (r = .62) among obese subjects following the surgery-induced weight loss. Bariatric surgery and concomitant weight loss recover the interaction between MOR and D2R in the ventral striatum in the morbidly obese. Consequently, the dysfunctional opioid-dopamine interaction in the ventral striatum is likely associated with an obese phenotype and may mediate excessive energy uptake. Striatal opioid-dopamine interaction provides a feasible target for pharmacological and behavioral interventions for treating obesity.Obesity is a growing burden to health and the economy worldwide. Obesity is associated with central µ-opioid receptor (MOR) downregulation and disruption of the interaction between MOR and dopamine D Previous studies indicate that both opioid and dopamine systems in the brain’s reward circuit are dysfunctional in obesity. The endogenous opioid system has been linked to hedonic aspects of feeding in animals4. In humans, both µ-opioid receptor (MOR) antagonists and inverse agonists have been shown to reduce eating behavior6. Previously, decreased MOR availability has been observed in the reward circuit among obese subjects8. Thus, aberrant opioid functioning in obesity may diminish the opioid-dependent rewarding effects of eating. Alterations in dopamine D2 receptor (D2R) expression and function in obesity have been observed in some12 but not all human imaging studies13. It is possible that the relationship between D2R availability and measures of obesity is not linear, but quadratic14 or age-dependent15. Alternatively, it is possible that the effects of obesity on D2R are mediated via the MOR system.The prevalence of obesity is dramatically increasing and there is an urgent need for novel efficient therapies. Numerous studies point towards the role of the brain in the development and maintenance of obesity16, but only a few studies have actually investigated this issue. In humans, dopamine-releasing drugs such as cocaine and amphetamine lead to endogenous opioid release19. In rats, both D2Rs and MORs are closely connected in the striatum, which can be morphologically divided into striosome/patch and matrix compartments. MORs can control the release of dopamine by inhibiting GABAergic interneurons in VTA25. Furthermore, VTA dopamine neurons express MOR postsynaptically, and direct inhibition between MOR and dopamine neurons exists without GABAergic signaling26.Tight interaction between dopaminergic and opioidergic systems has been proposed to underlie human reward functions27, while MOR antagonists block dopamine release and reduce food consumption28. Finally, in vivo PET data from humans show that there is a close interaction between MOR and D2R receptors in the reward circuit among non-obese subjects, while this interaction is disrupted in the ventral striatum among obese subjects, potentially contributing to obesity29. However, it remains unclear whether the dysfunctional MOR/D2R interaction reflects a vulnerability endophenotype for obesity, or whether it develops as a consequence of the obese state.Cross-talk between opioidergic and dopaminergic systems may underlie aberrant reward-related behaviors, such as excessive feeding. In rats, intravenous administration of MOR agonists triggers dopamine release and feeding30. The surgical procedure significantly lowers appetite31, but the actual molecular brain mechanisms behind this are still poorly understood. Bariatric surgery provides a powerful method for investigating changes in neuroreceptor systems and opioid-dopamine interaction after weight gain. Previous studies have investigated the effects of bariatric surgery and following weight loss to separate receptor systems, showing mainly unaltered D2R availability and normalized MOR availability35. Here we tested whether bariatric surgery-induced weight loss could recover the dysfunctional opioid-dopamine interaction in the obese.Bariatric surgery is the most effective method for weight loss in obesityhttp://www.clinicaltrials.gov). All participants gave a signed informed consent form prior to scans.The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Committee of the Hospital District of South-Western Finland  eligible for bariatric surgery. Either Roux-en-Y gastric bypass or sleeve gastrectomy was performed as their standard clinical treatment. Four subjects discontinued the study for personal reasons, and 21 subjects participated in the postoperative scanning six months after the surgical procedure. 20 non-obese healthy women (mean BMI 22 kg/m2) formed the control group. Data for this patient cohort have been reported previously34. The sample size was determined by a priori power analysis based on our previous studies34. Characteristics of the subjects are presented in Table We recruited 25 morbidly obese women (mean BMI 41 kg/m2 receptor availability with the antagonist [11C]raclopride36 and µ-opioid receptor availability with the high-affinity agonist [11C]carfentanil37 using positron emission tomography (PET) on two separate visits. Subjects were scanned again with both radiotracers six months after bariatric surgery. Radiotracer production has been described previously8. Both radioligands had high radiochemical purity (>99%). Before scanning, a catheter was placed in the subject’s left antecubital vein for tracer administration. The Head was strapped to the scanner table in order to prevent head movement. Subjects fasted two hours prior to scanning. A CT scan was performed to serve as an attenuation map. The clinical well-being of subjects was monitored during the scanning.We measured D11C]carfentanil and [11C]raclopride are presented in Table TM 690 PET/CT scanner  for 51 min, using 13-time frames. MR imaging was performed with Philips Gyroscan Intera 1.5T CV Nova Dual scanner to exclude structural abnormalities and to provide anatomical reference images for the PET scans. Anatomical images (1 mm3 voxel size) were acquired using a T1-weighted sequence .We injected both tracers as a bolus in separate scans on separate days. Injected amounts of 1Ccarfentwww.fil.ion.ucl.ac.uk/spm/) running on Matlab R2012a . To correct for head motion, dynamic PET images were first realigned frame-to-frame. The individual T1-weighted MR images were coregistered to the summation images calculated from the realigned frames. Regions of interest (ROIs) for reference regions were drawn manually on MRI images using PMOD 3.4 software . The occipital cortex was used as the reference region for [11C]carfentanil and cerebellum for [11C]raclopride. Receptor availability was expressed in terms of BPND, which is the ratio of specific to non-displaceable binding in the brain. BPND was calculated by applying the basis function method for each voxel using the simplified reference tissue model (SRTM) with reference tissue time-activity curves (TAC) as input data38.All alignment and coregistration steps were performed using SPM8 software . Preoperative differences in striatal volumes between obese and non-obese subjects were quantified with independent samples t-test and after bariatric surgery in the obese subjects using repeated-measures 2R availabilities are presented in the supplementary table  and dorsal caudate  in the control subjects , but the unaltered association in dorsal caudate   among obese subjects following the surgery-induced weight loss   and substantia nigra, whereas neurons projecting to GABAergic neurons are distributed in the matrix compartment45. Pathways projecting from striosomes back to the midbrain exert disinhibitory control over the dopaminergic neurons46, thus having a direct influence on the reward functions. These neurons are under direct opioidergic control47. Accordingly, endogenous opioids disinhibit the neurons projecting from the patches to the midbrain , and in this way increase dopaminergic firing in VTA. The rewarding effects of opioids are dependent on the MORs located in the striosomes48MORs are co-localized with D48, we hypothesize that aberrant opioid function in obese humans might lead to diminished dopamine release caused by eating. When obese subjects lose weight, the interaction between MOR and D2R is reverted. This further supports the notion that the interaction between these receptor systems is a normal state. The dysfunction of opioid-dopamine interaction in the ventral striatum might be an important factor underlying overeating, and thus a feasible target for pharmacological and behavioral interventions. This has already been noted in pharmacological studies. MOR antagonist naltrexone therapy alone does not lead to significant weight loss, but promising results are obtained when it is coupled with bupropion (a dopamine and norepinephrine reuptake inhibitor)52. Combination therapy of naltrexone and bupropion has been approved by FDA and EMA for weight management in adults53 and a certain amount of obese patients achieve significant weight loss56. The favorable effect of the combination therapy may be due to the tight coupling of MOR and D2R. Moreover, the better efficacy of the combination therapy over monotherapies underlines the complex pattern of neurotransmitter networks underlying overeating and suggests that both aspects of reward functions—wanting and liking, processes mediated by dopaminergic and opioidergic systems, respectively57—have to be taken care of in order to treat obese patients.Based on the observation that dopamine release caused by opioids in the striatum is dependent on the MORs in the striosomes in miceThis study has certain limitations. Only female subjects were studied, and the results may not be generalizable to male subjects. It was not possible to differentiate the combined effects of postoperative weight loss and altered gut anatomy and function. Altered neuroreceptor interaction may be due to the changes in gut hormones but also due to reduced intake of palatable foods. Further studies are needed to elucidate the sole effect of weight loss due to altered energy intake on the interaction of opioid and dopamine receptors by comparing the effects of weight loss by surgery versus dieting.Obesity is associated with disrupted opioid-dopamine interaction in the ventral striatum, but this is recovered by weight loss after bariatric surgery. The dysfunction of opioid-dopamine interaction might be an important factor underlying overeating.S1 Supplementary material


To compare the difference and agreement in central corneal thickness (CCT), keratometry (K), anterior chamber depth (ACD), aqueous depth (AQD), and lens thickness (LT) measured with CASIA 2 and IOLMaster 700 in patients with cataract.f, anterior Ks, real Kf, real Ks, ACD, AQD, and LT were measured with CASIA 2 and IOLMaster 700. Paired t-test, intraclass correlation coefficients (ICCs), 95% limit of agreement (95% LoA), and Bland-Altman plots were performed and used to analyze the difference and agreement between the two devices.A total of 81 patients with cataract (81 eyes) scheduled for phacoemulsification were prospectively collected from March to May, 2020 in the cataract department of Zhongshan Ophthalmic Center, Sun Yat-sen University, including 43 males and 38 females with age of 61.5 ± 10.6 years. CCT, anterior KP = 0.483). Differences among the CCT, anterior Kf, real Kf, real Ks, ACD, AQD, and LT measured by the two instruments were statistically significant (P < 0.001). The ICCs of CCT, anterior Kf, anterior Ks, real Kf, real Ks, ACD, AQD, and LT measurements between the two devices were 0.892, 0.991, 0.991, 0.827, 0.817, 0.937, 0.926, and 0.997, respectively. The 95% LoA between CASIA 2 and IOLMaster 700 was −30.06 to 0.43 μm for CCT, −0.3 to 0.48 D for anterior Kf, −0.46 to −0.43 D for anterior Ks, −1.49 to −0.49 D for real Kf, −1.62 to −0.49 D for Real Ks, −0.03 to 0.24 mm for ACD, 0.04 to 0.25 mm for AQD, and −0.06 to 0.09 mm for LT.There was no statistically significant difference in anterior K measurement with the CASIA 2 (44.3 ± 1.66 mm) and IOLMaster 700 (44.31 ± 1.67 mm, f, anterior Ks, ACD, AQD, and LT have excellent agreement between the two devices. CCT, real Kf, and real Ks have moderate agreement between the two devices. It is recommended to use anterior Kf, anterior Ks, ACD, AQD, and LT interchangeably between CASIA 2 and IOLMaster 700.Anterior K At present, modern cataract surgery has shifted from restoring vision to refractive surgery. Surgeons need to customize the refractive prediction of different patient to meet their visual expectations. Therefore, the accuracy of intraocular lens (IOL) calculation formula is very important. Olsen T pointed out that keratometry (K) measurement error accounted for 22% of total prediction error, and that ACD measurement error accounted for 42% , 2. TherAt present, many methods of measuring anterior segment parameters are used in clinical practice; however, the same parameter measured by different instruments often has systematic deviation. Previous studies have compared the agreement of IOLMaster 700 with IOLMaster 500 , LenstarCASIA 2 , the second generation of anterior segment OCT has emerged in recent years, combines Fourier domain technology and frequency sweep source OCT technology to further optimize scanning speed , scanning depth (16 × 16 × 13 mm), scanning density, and imaging resolution. However, its measurement accuracy remains unclear –15. As wIn this study, we aimed to compare the difference and agreement between CASIA 2 and IOLMaster 700 in anterior segment parameter measurements.Eighty one patients with cataract (81 right eyes) scheduled for phacoemulsification at Zhongshan Ophthalmic Center of Sun Yat-sen University from March 2020 to May 2020 were enrolled in this study, including 43 males and 38 females, aged 61.5 ± 10.6 years. Exclusion criteria were as follows: patients with dry eye, keratitis, pterygium, corneal scar, keratoconus and other ocular surface diseases; glaucoma, uveitis, retinal detachment, and other intraocular diseases, nystagmus leading to failure of fixed vision, and those who had been wearing contact lenses for a long time, and those who had a history of ocular trauma and eye surgery were excluded. This study was in line with the declaration of Helsinki and was approved by the ethics committee of Zhongshan Ophthalmic Center. All the patients signed informed consent.f, anterior Ks, real Kf, real Ks, ACD, AQD, and LT.Anterior segment measurements with CASIA2 and IOLMaster 700 were performed in a same dark room by two examiners. After 30 min of mydriasis with 1% tropicamide, the patients were asked to sit in front of the equipment in the dark room. After a complete blink, the patients should focus on the cursor in the instrument and open their eyes as much as possible to complete a measurement. For CASIA2, the examiners evaluated image quality during inspection and selected the results with acceptable quality for statistical analysis. For IOL Master700, images with high quality evaluated by built-in software were enrolled for analysis. All the parameters involved in this study measured with CASIA2 and IOLMaster 700 were automatically measured with the built-in software. Each participant has one measurement by CASIA2 and IOLMaster 700. The parameters we analyzed in this study include CCT, anterior Kt-test was performed to analyze the difference of measurement data that conform to normal distribution, and rank sum test is performed for measurement data that do not conform to normal distribution. Intraclass correlation coefficients (ICCs) and Bland-Altman plots were used to assess the agreement of the two devices. ICCs are a widely used reliability index in reliability and agreement analyses. This index ranges between 0 and 1, with values closer to 1 representing stronger reliability or agreement  was used to analyze the right eye data of all the patients. All continuous variables were expressed using mean ± standard deviation. Paired greement . ICCs weA total of 81 patients (81 right eyes) with an average age of 61.5 ± 10.6 years were included in this study. t-test. The average difference in anterior Kf was 0.09 ± 0.2 D; that in anterior Ks was −0.02 ± 0.23 D; that in real Kf was −0.99 ± 0.25 D; that in real Ks was −1.06 ± 0.29 D; that in CCT was −14.81 ± 7.78 mm; that in ACD was 0.13 ± 0.05 mm; and that in AQD was 0.15± 0.05 mm. Except for anterior Ks (P = 0.483 >0.05), differences in the other parameters were statistically significant (P < 0.001).The measurement results with CASIA 2 and IOLMaster 700 are shown in f, anterior Ks, real Kf, real Ks, ACD, AQD, and LT were 0.892, 0.991, 0.991, 0.827, 0.817, 0.937, 0.926, and 0.997, respectively. The ICC showed that CASIA 2 had a good agreement with IOLMaster 700 in anterior Kf, anterior Ks, ACD, AQD, and LT. Also, there was a moderate agreement between the two devices in the measurement of CCT, real Kf, and real Ks.The ICC of CCT, anterior Kf, anterior Ks, ACD, AQD, and LT measurements between CASIA 2 and IOLMaster 700 had a narrow 95% LoA of 0.3 to 0.48 D, −0.46 to 0.43 D, 0.03 to 0.24 mm, 0.04 to 0.25 mm, and −0.06 to 0.09 mm, respectively, indicating a good agreement in those parameters. The CCT, real Kf, and real Ks measurements between the two instruments had a little bit broad 95% LoA of −30.06 to 0.43 μm, −1.49 to −0.49 D, and −1.62 to −0.49 D, respectively, indicating a moderate agreement in those parameters.f, anterior Ks, real Kf, real Ks, CCT, ACD, AQD, and LT. The comprehensive analysis results showed that there was a good agreement in the anterior Kf, anterior Ks, ACD, AQD, and LT measurements and a moderate agreement in the CCT, real Kf and real Ks measurements.This study analyzed the agreement between CASIA 2 and IOLMaster 700 in anterior Kf and anterior KS. The ICC results showed that there were high agreements in the measurements of anterior Kf , anterior Ks , ACD , AQD , and LT , and moderate agreements in the measurements of CCT , real Kf , and real Ks .The agreement between the two instruments was high in terms of anterior Kf and anterior Ks were close between CASIA2 and IOLMaster700, while results of real Kf and real Ks showed significant difference between two machines. So anterior Kf and anterior Ks would be used interchangeably with CASIA2 and IOL Master700. In a previous study, Wylegala et al. compared CCT and anterior corneal curvature measured with CASIA 2, Galilei G6 , and RevoNX   . Their rf and real KS measured with CASIA 2 and IOLMaster 700 is may be due to the different principles of the two instruments. CASIA 2 takes 32 measuring points from the central 3-mm area of the cornea and connects the two points symmetrically centered on the corneal apex to form 16 straight lines. Among these 16 lines, the line with strongest diopter is Ks, and the radius of curvature in the direction with an angle of 90° with Ks is Kf. Using this principle, CASIA 2 can calculate the anterior and posterior corneal curvatures and the real corneal curvature by paraxial calculation using the Gullstrand model eye refractive index. The IOLMaster 700, based on frequency sweep technology, can only measure the corneal curvature of the anterior surface, and its measurement principle is to obtain eight measuring points from 2.5 mm in the center of the cornea. According to projection images of the eight measuring points, an ellipse is fitted. The short radius of the ellipse is K2, and the radius in the direction of 90° with K2 is considered as K1. Therefore, the difference in measurement principle may be the main reason for the difference in real Kf and real KS measurement results between CASIA 2 and IOLMaster 700. As suggested by previous studies, our results show that the parameters real Kf and real KS of the two instruments should not be used interchangeably.Our results show that the difference in real KThe agreement in ACD, AQD, and LT measured with CASIA 2 and IOLMaster 700 was high and CCT measurement had a moderate agreement. CASIA 2 has a different agreement with CCT, ACD, AQD, and LT measured by other instruments. Previous study has shown a high correlation of measurements result of ACD, anterior chamber width and other parameter between CASIA2 and time-domain AS-OCT , howeverCASIA 2 is also consistent with IOLMaster 700 in CCT, ACD, AQD, and LT, even if there are acceptable differences. The differences between both instruments in the measurement of CCT, ACD, AQD, and LT were possible due to different technology and different image analysis principles. CASIA 2 utilizes a wavelength of 1,310 nm, and its axial resolution is <10 μm , while IIn clinical application, CASIA2 is rather an OCT, which is mainly used to measure biological parameters of an ocular anterior segment, such as calculating the size of an ICL. IOLMaster 700 is a biometer, which is mainly used to measure axial length and calculate IOL power. The anterior and posterior of the cornea are crucial to patients who will have an implant of a trifocal lens. The posterior corneal surface curvature with CASIA2 is measured by simulation. The type of IOL Master 700 we used in this study cannot measure posterior corneal surface curvature. The new type of IOL Master, 700 TK, can measure posterior corneal surface curvature directly. Currently, the golden standard in cornea measurement is Pentacam. There are few studies on agreement between CASIA2 and Pentacam that showed the agreement in anterior and posterior corneal curvature is acceptable . There iThere are several limitations in this study. In this study, we included only eyes without a history of underlying eye diseases and eye surgery. We believe that the agreement of measurement results is likely to be lower for patients with underlying eye diseases . Second, only patients with cataract were included in this study. More multicenter clinical trials are needed to verify the validity and reliability of CASIA 2 in different populations. At the same time, both CASIA 2 and IOLMaster 700 are optical imaging OCTs, which will cause geometric optical distortion in the imaging process. The correction methods of different manufacturers may be inconsistent. However, due to the protection of patent law, we cannot know the specific correction scheme.f, anterior Ks, ACD, AQD, and LT between the instruments interchangeably. In addition, CASIA 2, with high penetrability and high resolution, is more valuable for the diagnosis and treatment of ophthalmic diseases.Our study compared CASIA 2 with IOLMaster 700. It is recommended to use anterior KThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Zhongshan Ophthalmic Center. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.ZL and LL conceived and designed the research. GY, XR, XG, and JZ collected the data. ZX, GY, and XR analyzed the data. GY and XR wrote the manuscript. LL and ZX critically revised the manuscript. All authors discussed the results and provided comments on the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (81873675 and 81770905) and the Construction Project of High-Level Hospitals in Guangdong Province (303020102).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The interaction between gas flow and liquid flow, governed by fluid dynamic principles, is of substantial importance in both fundamental science and practical applications. For instance, a precisely designed gas shearing on liquid solution may lead to efficacious production of advanced nanomaterials. Here, we devised a needleless Kármán vortex solution blow spinning system that uses a roll-to-roll nylon thread to deliver spinning solution, coupled with vertically blowing airflow to draw high-quality nanofibers with large throughput. A wide variety of nanofibers including polymers, carbon, ceramics, and composites with tunable diameters were fabricated at ultrahigh rates. The system can be further upgraded from single thread to multiple parallel threads and to the meshes, boosting the production of nanofibers to kilogram scale without compromising their quality. A needleless solution blow spinning system based on Kármán vortex is developed for high-throughput production of nanofibers. Nanofibers enjoy the luxury of being one of the most attractive nanomaterials because of their unique physicochemical properties and characteristics  clarified an unusual alternating stream of vortices that resulted from fluid flowing past a cylinder. This ubiquitous phenomenon, now known as a Kármán vortex (KV) street, can be found at all scales of fluid motion (fig. S1)  solution was used as an example to understand the spinning process and mechanism]. The spinning system was assembled by a roller, a vessel filled with spinning solution, a loop-locked nylon thread, and a gas pipeline set vertically to the thread to provide high-speed air blowing. The spinning solution was continuously carried out via a circulating nylon thread passing through the vessel. The nylon thread was wrapped with spinning solution because of its specific viscosity and a certain degree of wettability with nylon (fig. S2). Driven by the high-speed airflow, the solution was shaped into a Taylor cone and ejected at a high speed, forming nanofibers via fast stretching and swinging, followed by the evaporation of the solvents  simulations, and bead-spring model for simulating the fiber behavior  . The gas shearing on liquid solution jet holds the key to the formation of uniform fibers with thinner diameters. We emphasize that a large velocity gradient formed on the leeward side of the thread . This grQ criterion .2O, PS/DMF, PVA/H2O, and PVP/ethanol (EtOH)] were highly stable , were fabricated by postprocessing corresponding precursor fibers, showing potential for applications in high-performance catalyst, sensor, structure reinforcement, and thermal insulation , polyvinyl pyrrolidone (PVP), polyvinyl butyral (PVB), polyimide (PI), polyvinylidene fluoride (PVDF), cellulose acetate (CA), polyethylene oxide (PEO), and polystyrene (PS) as well as carbon, ceramics, and composite nanofibers . The spi2 nanoparticle-loaded PAN nanofibers (SiO2 NPs @ PAN) by KV-SBS  %], the solution failed to form a jet under gas shearing, and the formation of tiny individual droplets instead of fibers was observed (fig. S20A). However, at much higher concentrations , the polymer chain got entangled, leading to insufficient jet stretching and poor fiber quality (fig. S20D). At moderate concentrations , the solution jet experienced a linear motion for a moment before starting to oscillate downstream .We investigated the effect of the thread speed and the diameter of the gas pipeline . Then, we fixed the thread speed to 2.5 cm/s to explore the effect of solution concentration on fiber diameter and production rate . The results showed that higher concentrations resulted in the formation of fibers with larger diameters (figs. S21C and S22). The production rate first increased and then decreased as the solution concentration increased, reaching a peak value of 5.90 ± 0.19 g/hour per thread at a concentration of 16 wt % (fig. S21D). This is consistent with our observations made by the high-speed camera: At high concentrations, it is difficult to elongate the polymer solution because of its high viscosity. The production rates of KV-SBS system were found to be higher than those of many traditional and/or modified electrospinning methods  = 250,000] was dissolved into 44 g of DMF (99.5%) to obtain PAN/DMF solution. Six grams of PVA (PVA-1788) was dissolved into 44 g of deionized water to obtain PVA/H2O solution. Six grams of PEO  was dissolved into 44 g of deionized water to obtain PEO/H2O solution. Six grams of PVP  was dissolved into 44 g of ethanol to obtain PVP/EtOH solution. Five grams of PVB  was dissolved into 45 g of ethanol to obtain PVB/EtOH solution. Four grams of PVDF  was dissolved into 46 g of DMF to obtain PVDF/DMF solution. A total of 7.5 g of PS  was dissolved into 42.5 g of DMF to obtain PS/DMF solution. Ten grams of PMMA  was dissolved into 40 g of DMF to obtain PMMA/DMF solution. Six grams of CA  and 0.5 g of PEO were dissolved into 44 g of DMF to CA/DMF solution. The polymer solutions were poured into the vessel and spun into corresponding nanofibers, respectively. As for PI nanofibers, a quantity of 0.2 g of PEO was first dissolved into 20 g of DMF. Then, 20 g of polyamide acid (PAA)/DMF solution (20 wt %) was added to the previously prepared PEO/DMF solution. The mixed solution was magnetically stirred at 50°C for 6 hours to obtain a homogeneous solution. Successively, PAA nanofibers were obtained via the KV-SBS process. Subsequently, the PAA nanofibers were treated at 300°C by setting the heating rate to 5°C min−1 and by using a holding time of 1 hour. Last, PI nanofibers were obtained.Six grams of PAN  was added to the solution. The solution was magnetically stirred for 6 hours at room temperature. Afterward, the homogeneous solution was used to prepare AlCl3/PVP nanofiber sponge via the KV-SBS process. The nanofiber sponge was immediately treated at 1100°C for 2 hours with a heating rate of 5°C min−1 in the air before cooled down in the furnace. Last, the Al2O3 nanofibers were obtained.Four grams of PVP was dissolved into a mixture of 20 g of deionized water and 20 g of ethanol. Then, 12 g of aluminum chloride hexahydrate [AlCl2·8H2O, AR) was added to the solution. The solution was magnetically stirred for 6 hours at room temperature. Afterward, the homogeneous solution was used to prepare ZrOCl2/PVP nanofiber sponge via the KV-SBS process. The nanofiber sponge was immediately treated at 800°C for 2 hours with a heating rate of 5°C min−1 in the air before cooled down in the furnace. Last, the ZrO2 nanofibers were obtained.Four grams of PVP was dissolved into a mixture of 20 g of deionized water and 20 g of ethanol. Then, 8 g of zirconium oxychloride octahydrate 4/PVP nanofiber sponge via the KV-SBS process. The nanofiber sponge was immediately treated at 450°C for 2 hours with a heating rate of 5°C min−1 in the air before cooled down in the furnace. Last, the TiO2 nanofibers were obtained.Four grams of PVP was dissolved into a mixture of 20 g of deionized water and 20 g of ethanol. Then, 16 g of tetrabutyl titanate [Ti(OBu)2 nanoparticles (30 nm) was added to the solution. The solution was magnetically stirred for 6 hours at room temperature. Afterward, the homogeneous solution was used to prepare SiO2 NPs @ PAN nanofibers via the KV-SBS process.Six grams of PAN was dissolved into 44 g of DMF to obtain 12 wt % PAN/DMF solution. Then, 3 g of SiOPAN/DMF solution (12 wt %) and PVDF/DMF solution (8 wt %) were poured into two vessels, respectively. Then, they were spun into nanofibers at the same time (see the schematic diagram in fig. S18A), and PVDF/PAN nanofiber composites were obtained.Ten grams of PS was dissolved into 40 g of DMF to obtain 20 wt % PS/DMF solution. Afterward, the solution was used to prepare PS nanofibers via the KV-SBS process. A cage-like porous collector was used to collect nanofibers as a sponge.3PO4, and 14.4 g of AlCl3·6H2O were subsequently added to the PVA solution. The solution was magnetically stirred for 6 hours at room temperature. Afterward, the homogeneous solution was used to prepare the precursor fiber mat via the KV-SBS process. The mat was immediately treated at 1000°C for 2 hours with a heating rate of 5°C min−1 in the air before cooled down in the furnace. Last, the SiO2-Al2O3 composite ceramic fiber mat was obtained.A total of 4.5 g of PVA powder was dissolved into 43.2 g of deionized water. Then, 15.6 g of TEOS, 0.08 g of H2, Al2O3, ZrO2, and TiO2 nanofibers were detected by x-ray diffraction  test, where the x-ray was Cu-Kα radiation, the scanning speed was 5° min−1, and the scanning range was 10° to 80°.The morphological properties and elementary composition of nanofibers were measured by a field-emission scanning electron microscope  equipped with energy-dispersive x-ray spectroscopy. The average fiber diameter was obtained by analyzing 200 U using Image-Pro Plus software . Crystal structure of the carbon and SiOA high-speed camera  equipped with an F-mount lens  was used for the direct observation of the KV-SBS process. A 100-W light-emitting diode lamp  was used to continuously illuminate the spinning area, which enabled the acquisition of clear images with a short exposure time of the high-speed camera. All the high-speed images were captured at a frame rate of 8000 frames per second (fps), with a picture resolution of 1920 × 416 pixels and an exposure time of 60 μs. The analysis of the videos obtained was carried out using Motion Studio software. All the high-speed videos are played at a frame rate of 24 fps using the images obtained at 8000 fps.The PS nanofiber sponges were placed in solvents until they were filled with the organic liquid. Then, they were taken out for weight measurement quickly. Here, the sorption capacity was evaluated by weight gain, which is the ratio of the mass of absorbed organics to that of the nanofiber sponge.


We find that increasing and decreasing DA biases striatal activity toward the direct and indirect pathways, respectively, by changing the overall number of SPNs recruited during behavior in a manner not predicted by existing models of DA function. This modulation is drastically altered in a model of Parkinson’s disease. Our results reveal a previously unappreciated population-level influence of DA on striatal output and provide novel insights into the pathophysiology of Parkinson’s disease.Dopamine (DA) is a critical modulator of brain circuits that control voluntary movements, but our understanding of its influence on the activity of target neurons in vivo remains limited. Here, we use two-photon Ca The neuromodulator dopamine (DA) is an essential component of basal ganglia circuits that control goal-directed behaviors. Considerable evidence in humans, primates, and rodents implicates DA in supporting motor learning and in executing vigorous movements via its actions in the striatum, the principal input nucleus to the basal ganglia . The strs-coupled D1-type DA receptors and iSPNs Gαi-coupled D2-type DA receptors, DA is widely believed to differentially modulate both pathways and to promote imbalances that impact how the basal ganglia contribute to behavior , in which dSPNs are selectively labeled red  or virally . We refer to this cohort as tdTomatoAdora2a mice henceforth. Ca2+ signals obtained from identified iSPNs were comparable to those imaged in putative iSPNs in tdTomatoDrd1a mice  to directly image Ca2+ signals in cholinergic interneurons  in tdTomatoDrd1a and tdTomatoAdora2a mice. Under these conditions, intracellular Ca2+ transients are unlikely to reflect individual spikes, but rather bursts of action potentials (2+ transients in dSPNs and iSPNs was exceedingly low during periods of immobility (treadmill speed < 0.2 cm/s) and rose sharply at movement onset without notable temporal offset between pathways  16.9 ± 1.8% and 18.6 ± 1.8% of all imaged dSPNs and iSPNs, respectively  – is balanced, with few notable differences distinguishing dSPNs from iSPNs during self-paced forward locomotion.SPNs showing Cansembles . We quanectively . For eaclocities , consistlocities . Importalocities . Ensembllocities . Thus, t2+ signals in dSPNs and iSPNs before and after systemic administrations of a cocktail of D1 and D2-type dopamine receptor (D1/2R) antagonists (0.2 mg/kg SCH23390 and 1 mg/kg raclopride). Although this manipulation did not impact the velocity or duration of locomotor bouts on the treadmill, it curtailed the total number of bouts that mice spontaneously initiate compared to vehicle-treated animals , resulting in a strong imbalance in favor of the indirect pathway  while mice locomoted on motorized treadmills to mitigate effects on spontaneous behavior  in comparison to vehicle treatment.Given the marked effects of Densitive . To testbehavior . We exam−5, Kruskal-Wallis test; 1/2R agonists promoted a striking decline in the prevalence of both active dSPNs and iSPNs, with iSPNs showing the strongest decrease. The drop in the number of active iSPNs suggest that iSPNs are not maximally repressed by baseline DA tone in vivo  inhibition. Presynaptic reuptake of DA into axonal terminals is the primary mechanism through which DA neurons limit the amplitude and duration of DA transients in the striatum, and inhibiting DAT leads to strong and prolonged elevations in extracellular DA in striatum . We thertrations , as nomitrations . These dThe degeneration of substantia nigra pars compacta (SNc) DA neurons in animal models of Parkinson’s disease evokes imbalances in activity rates in dSPNs and iSPNs . To exam2,53 = 3.66, p=0.03; 2,53 = 11.92, p=5.3×10−5; 2,53 = 19.0, p=6.1×10−7; 2+ transients was halved, whereas active iSPNs within the same FOV more than doubled in number. These effects, which were similar to those observed with DA receptor antagonists  are shared between pathways and not subject to strong pathway-specific modulation by DA. This finding stands in contrast to a recent imaging study reporting differential changes in the rate of Ca2+ events in dSPNs and iSPNs upon acute and chronic DA manipulations  and ChATCre knock-in mice (stock #: 031661) were purchased from The Jackson Laboratory and bred with C57BL/6J wild type mice (stock #: 000664). Transgenic mice expressing Cre selectively in iSPNs  or dSPNs   encoding Cre-dependent tdTomato . Experiments were carried out using both male and female mice heterozygous for all transgenes at 8–24 weeks of age.All procedures were performed in accordance with protocols approved by the NYU Langone Health Institutional Animal Care and Use Committee (protocol # 170123). Mice were housed in group before surgery and singly after surgery under a reverse 12 hr light-dark cycle (dark from 10 a.m. to 10 p.m.) with ad libitum access to food and water. ; EY217)  were genMice were administered dexamethasone  1–2 hr prior to surgery. They were then anaesthetized with isoflurane, placed in a stereotaxic apparatus (Kopf Instruments) on a heating blanket (Harvard Apparatus) and administered Ketoprofen . The scalp was shaved and cleaned with ethanol and iodine solutions before exposing the skull. A custom titanium headpost was implanted over lambda using C and B metabond (Parkell) to allow head fixation. To achieve widespread viral expression of GCaMP6f in striatum, 100 nl of AAV1-Syn-GCaMP6f-WPRE-SV40 was injected alone or along with AAV1-FLEX-tdTomato 1.7 mm below dura at a rate of 100 nl/min (KD Scientific) into the right dorsolateral striatum at four locations : 0.7/1.7; 0.7/2.3; 1.3/1.7, and 1.3/2.3. Injection pipettes were left in place for 5 min before removal. A 3 mm craniotomy was then drilled  and cortical tissue was aspirated until the corpus callosum lying above the striatum was exposed, as described previously . A custoFor experiments characterizing the effects of SNc neuron loss on striatal activity, a second stereotaxic surgery was performed under isoflurane anesthesia after baseline imaging sessions. Desipramine (25 mg/kg) and pargyline (5 mg/kg) were administered intraperitoneally prior to surgery to increase the selectivity and efficacy of 6-OHDA lesions . A smallMice were deeply anesthetized with isoflurane and perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer. Brains were post-fixed for 1–3 days, sectioned coronally (50–100 μm in thickness) using a vibratome  and processed for immunofluorescence staining for tyrosine hydroxylase  and dopamine transporter  using standard methods. Brain sections were mounted on superfrost slides and coverslipped with ProLong antifade reagent with DAPI (Molecular Probes). Endogenous tdTomato and GCaMP6 fluorescence were not immuno-enhanced. Whole sections were imaged with an Olympus VS120 slide scanning microscope and high-resolution images of regions of interest were subsequently acquired with a Zeiss LSM 800 confocal microscope. TH and DAT immunofluorescence were quantified in ImageJ (NIH) by measuring mean pixel intensity in similarly sized regions of interest in VTA, SNc, dorsal striatum and ventral striatum in both intact and lesioned hemispheres. After subtracting background signal from adjacent unstained brain regions, fluorescence intensity values in the lesioned hemisphere were expressed as a percentage of values measured on the intact side. Three measurements were obtained from each region of interest along the anterior-posterior axis and averaged together for each mouse.Drugs  were reconstituted and stored according to the manufacturers’ recommendations. 6-Hydroxydopamine  was dissolved freshly into sterile 0.9% NaCl and 0.2% ascorbic acid immediately prior to administration to minimize oxidation. Working concentration aliquots of desipramine (25 mg/kg), pargyline , S(-)raclopride , SCH23390 , nomifensine (10 mg/kg), (-)quinpirole (0.3–6 mg/kg), SKF81297 (0.3–6 mg/kg), and L-DOPA (10 mg/kg) were prepared daily in sterile physiological saline and administered intraperitoneally at minimum 10 min prior to experimentation. L-DOPA was co-administered along with the peripheral DOPA decarboxylase inhibitor benserazide hydrochloride .https://www.janelia.org/open-science/wavesurfer) to synchronize imaging and behavioral data. Striatal fields of view  near the center of the imaging window were selected based on the expression of GCaMP6f and tdTomato, and were continuously imaged with a resolution of 512 × 512 pixels at 30 Hz frame rate for a minimum of 20 min each. Bleaching of GCaMP6f was negligible. The same fields of view were imaged multiple times on non-consecutive days using alignment based on blood vessels and a reference image.Imaging was performed through a 20X long working-distance air objective  on a galvo-resonant scanning microscope  equipped with GaAsP photomultiplier tubes and under the control of ScanImage five software (Vidrio Technologies). GCaMP6f and tdTomato were excited a using a pulsed dispersion-compensated Ti:Sapphire laser  tuned to 940 nm (40–80 mW at the sample). Mice were head-fixed above a freely rotating circular treadmill consisting of a 6’ plastic saucer (Ware Manufacturing) mounted on a rotary encoder . Motorized, fixed-distance and velocity trials were conducted by connecting a integrated servo motor (Teknic Clearpath: CPM-MCVC-2310S-RQN) to the treadmill to impose 10 s-long fixed-speed (10 cm/s) running bouts every 20 s. Water-rewards (~0.1 ml) were delivered through a spout controlled by an inaudible solenoid  at 60 s intervals between motorized running bouts. Mouse posture was simultaneously monitored using a near infrared camera , with each video frame triggered by the two-photon imaging frame clock. The latter was recorded in Wavesurfer  was passed through a fluorescence mini-cube  and 400 μm fiber optic patch cord connected to the mouse via a zirconia sleeve. Emission was collected through the same patch cord and mini-cube to a femtowatt photoreceiver . Photometry signals were digitized at 2 kHz using a National Instruments acquisition board (USB-6343), recorded with Wavesurfer, and low-pass filtered and down-sampled to 30 Hz in MATLAB (Mathworks).2+ fluorescence traces were extracted from individual neurons with minimal neuropil contamination using a custom MATLAB (Mathworks) pipeline described in detail in https://github.com/HarveyLab/Acquisition2P_class.git; Drd1atdTomato and Adora2atdTomato mice, respectively, and tdTomato-negative cells were either labeled as putative iSPNs (in in Drd1atdTomato mice), putative dSPNs (Adora2atdTomato mice), or putative interneurons based on cell morphology, baseline Ca2+ fluorescence intensity and Ca2+ transient kinetics . Ca2+ transients were detected using the built-in MATLAB findpeaks function on each cell’s neuropil-subtracted fluorescence trace smoothed with a 150 ms window if they exhibited a minimum peak height and minimum peak prominence of five standard deviations greater than baseline, and a minimum width of 140 ms at half peak prominence. These criteria are intentionally stringent so as to exclude events not clearly resolved from baseline fluorescence at the cost of underestimating Ca2+ transient frequency per SPN. All imaged neurons displaying at minimum one Ca2+ transient were deemed active. Individual fields of view were only selected for longitudinal imaging and quantification if at least five active dSPNs and five active iSPNs were observed at baseline in self-initiated and motorized locomotor trials, and if mice traveled at minimum five meters in self-initiated trials to ensure adequate estimation of SPN ensemble size and mean SPN Ca2+ transient frequency. For each imaging session and field of view (FOV), we report the percentage of dSPNs and iSPNs that display Ca2+ transients (i.e. active ensemble size), the frequency of Ca2+ transients recorded per active dSPNs or iSPNs while moving or immobile averaged across each FOV, and the mean amplitude of Ca2+ transients recorded in all active dSPNs or iSPNs averaged for each FOV. Active ensemble size is calculated as the percentage of all imaged dSPNs or iSPNs in a given FOV that display Ca2+ transients over the course of an imaging session. Activity in dSPNs and iSPNs was compared for each imaging session and FOV by computing a bias index, consisting of the difference in ensemble size, mean Ca2+ transient frequency or amplitude between dSPNs and iSPNs, divided by their sum. Treadmill velocity was extracted from positional information provided by the rotary encoder, down-sampled to 30 Hz and aligned to the two-photon imaging frame rate. Immobility is defined as any period of time beginning at least 0.5 s after treadmill velocity decreases below 0.2 cm/s, lasting at minimum 4 s, and ending 0.5 s before treadmill velocity exceeds 0.2 cm/s again and for which no postural movement is detected using the infrared camera. Movement bouts are defined as any period of time when absolute treadmill velocity exceeds 0.4 cm/s for a minimum of 4 s, and is preceded and followed by 4 s of immobility. Movement onsets and offsets are the first and last time points, respectively, at least two standard deviations away from treadmill velocity measured while immobile. To relate Ca2+ transient frequency to treadmill velocity, we recorded the instantaneous treadmill velocity at each Ca2+ transient onset and divided the total number of Ca2+ transients per velocity (binned at 0.4 cm/s) per SPN by the amount of time spent by a mouse at that velocity over the course of an imaging session. To relate the percentage of active SPNs to velocity, we averaged treadmill velocity in 200 ms bins, determined the fraction of all imaged dSPNs or iSPNs active within each bin and calculated the mean percentage of active dSPNs and iSPNs at different treadmill speeds (binned to 0.4 cm/s). To align Ca2+ transient frequency to movement onset/offset, we calculated the mean number of Ca2+ transients imaged per FOV per 200 ms-long bin around all locomotor bout onsets/offsets divided by the total number of active SPNs and bin duration.Quantification of imaging and behavioral data was carried out in MATLAB using custom-code available online  were collected at minimum from two separate cohorts of mice  and were compared in Prism 8 (Graphpad) using non-parametric Mann-Whitney U test for independent groups and Wilcoxon signed-rank test for matched samples, and Two-way ANOVAs (mixed-effect model) for multiple comparisons, as indicated in text and figure legends. Significant interactions were followed-up with within-group t-tests corrected for multiple comparisons (Bonferroni) between baseline and test data. p Values smaller than 0.05 were considered statistically significant and assigned the following nomenclature in Figures: *p<0.05, **p<0.01, ***p<0.005 and ****p<0.001. Reported n values represent the number of fields of view imaged from N mice. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:This paper is of broad interest to neuroscientists studying Parkinson's disease, dopamine (DA) modulation and the striatum. The study provides a unique view of how dopamine modulates striatal activity in vivo by investigating effects on the size of movement-related projection neuron ensembles in DA-intact and DA-depleted mice. Whereas prior studies have focused on DA modulation of SPN excitability and firing rates, this paper introduces highly novel concepts about how dopamine modulation of the number of activated neurons (ensemble size) may influence direct/indirect pathway balance and its potential significance for Parkinson's disease.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Gary Westbrook as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ilana B Witten (Reviewer #3). The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Thank you for submitting your article "Dopamine differentially modulates the size of projection neuron ensembles in the intact and dopamine-depleted striatum" for consideration by Essential revisions:As you can see in the comments below, there was great enthusiasm for your study by all three reviewers. The reviewers do not ask for new experiments, but please address the requests listed below to address because they will enhance clarity of presentation and interpretation. The full reviews are also included below for your consideration. For Reviewer 2 #3 item concerning evaluation of dyskinesia data, it is not required, but optional to address.1. Reviewer 1: Items 1-32. Reviewer 2: Items 1,2,4,5Reviewer #1 (Recommendations for the authors):The claims of the paper are largely well supported by current data. However, several aspects of data analysis could be done or clarified to further support the conclusions.1. The dSPN U-shaped curve response is a potentially novel finding, but appears to be supported by a single data set at 1mg/kg in Figure 4A. These data also includes values that are non-normally distributed and close to outlier-like values. Was this finding replicated in a second cohort?2. The authors focused on the analysis of ensemble size, firing rate and frequency of activated SPNs during forward locomotion. Presumably, locomotion arises from collaborative activation of certain SPN subsets and suppression of other subsets. Data in rearward locomotion and transitions should be readily available. Its evaluation would be important to bolster the findings, or reveal unexpected variances to the current interpretations.3. Similarly, when only "total locomotion" is presented as the outcome measure, sub analyses should be performed on richer features of the movement data and presented/discussed to ensure that differential findings are not equally or better explained by differences in forward, reverse, or stalled locomotion or velocity.Reviewer #2 (Recommendations for the authors):1. While D1 receptors, at least within the striatum, are relatively selective in their expression on dSPNs, D2 antagonists or agonists may act on iSPNs themselves, or local microcircuit elements, including cholinergic interneurons, dopamine terminals, and potentially local glutamatergic and GABAergic synapses. The authors allude to the glutamatergic inputs in the discussion, but one question in my mind was how much of the results here could be related to D2 autoreceptors on dopamine terminals. For example, D2 antagonists presynaptically increase striatal dopamine release, and D2 agonists decrease dopamine release . At the very least, it would be worth discussing the potential mechanisms of the pharmacology.2. Related to the above comment, though some of the differences between healthy and 6-OHDA treated animals in their response to dopamine drugs may be related to chronic changes in synaptic connectivity or intrinsic properties, some may relate to the loss of one of the targets of these drugs: the dopamine terminals. Might the effects of D2 antagonists in healthy animals be an interplay between direct effects on SPNs themselves, plus changes in dopamine release mediated by actions on dopamine terminals? And one of the reasons it's so different in 6-OHDA treated animals is that now this element has been removed?3. The authors refer to L-DOPA-induced dyskinesia in their discussion, and compare their findings to published work with L-DOPA-induced dyskinesia, but this behavioral phenomenon was not mentioned/quantified in the text. Based on my understanding of the literature, the severity of dopamine depletion they induced  combined with a dose of 10 mg/kg of L-DOPA would likely induce dyskinesia . Did their animals show dyskinesia? And if so, how did this relate to the patterns of neural activity reported?4. For interpretability and clarity, it would be helpful to include details of some major methods in the Results text and/or main Figure legends. For example, the authors should indicate in the Results and/or Figure legends that they used both an A2a-Cre x Ai14 cross and a viral approach to directly label iSPNs, that they used a cocktail of SCH23390 and raclopride to block D1/2 type dopamine receptors, or a cocktail of SKF81297 and quinpirole to activate D1/2 type dopamine receptors.5. It is unclear between the Figure and methods text what concentration of L-DOPA was used – the figure itself has "10" listed underneath , but the methods list 12 mg/kg (which may instead refer to benserazide dosing?).Reviewer #3 (Recommendations for the authors):I thought this was a nice study. There have been few studies to examine DA modulation of D1R and D2R activity and I thought the dual population imaging was cool, I was interested to see the large population level effects that mostly recapitulated expectations about the two pathways, as well as to see the inverted-U effect on D1Rs which was interesting and I was not aware of that being observed in striatum before. I didn't have any major concerns. Reviewer #1 (Recommendations for the authors):The claims of the paper are largely well supported by current data. However, several aspects of data analysis could be done or clarified to further support the conclusions.1. The dSPN U-shaped curve response is a potentially novel finding, but appears to be supported by a single data set at 1mg/kg in Figure 4A. These data also includes values that are non-normally distributed and close to outlier-like values. Was this finding replicated in a second cohort?Figure 4A consist of two independent cohorts of mice imaged 4 months apart and the extreme values belong to separate mice from each cohort. Even after excluding both extreme values from the dataset, treatment with D1/2R agonists still significantly increased dSPN ensemble size compared to baseline .It is indeed the case that the only dopamine receptor agonist dose tested that significantly increased dSPN ensemble size was the 1 mg/kg data set, and that two data points disproportionately contributed to its mean. Importantly, the data presented in However, we wish to correct the Reviewer on an important point: the 1 mg/kg agonist data point is not the only evidence for an inverted U-shaped distribution. First, we note that the fact that blocking and stimulating D1/2 receptors both reduce dSPN ensemble size is, in and of itself, supportive of an inverted U distribution, even in the absence of an overt increase in dSPN ensemble size with low dose agonists. Second, we provide evidence that elevating endogenous dopamine levels with natural rewards and dopamine transporter blockers respectively increases and decreases the size of dSPN ensembles , further supporting an inverted U-shaped relationship between dSPN ensemble size and dopamine signaling.2. The authors focused on the analysis of ensemble size, firing rate and frequency of activated SPNs during forward locomotion. Presumably, locomotion arises from collaborative activation of certain SPN subsets and suppression of other subsets. Data in rearward locomotion and transitions should be readily available. Its evaluation would be important to bolster the findings, or reveal unexpected variances to the current interpretations.Mice were habituated to head-fixation in the recording apparatus on both freely-rotating and motorized circular treadmills prior to experimentation. Once habituated, mice engaged in bouts of forward locomotion spontaneously on the freely-rotating treadmill and rarely (if ever) initiated rearward treadmill rotations. In fact, we interpreted rearward locomotion as an overt sign of behavioral distress, and thus prolonged habituation (and refrained from initiating imaging experiments) in mice that displayed such behavior. In addition, we never imposed rearward locomotion on the motorized treadmill. For these reasons, we do not have rearward locomotion data available for analysis.2+ event frequency and 5H for the percentage of imaged SPN active per 200 ms time bin. We also include in 2+ transients as mice transition between immobility and forward locomotion in the dopamine-intact and dopamine-depleted  striatum:We do, however, have data during transitions from immobility to forward locomotion and back to immobility. We report on these data in Figures 2B,C for Ca2+ event frequency per active SPN, we refrained from displaying them in the manuscript for clarity.These data are consistent with the changes in total SPN ensemble size reported in our manuscript. However, because measures of the instantaneous percentage of active SPNs cannot unambiguously distinguish between changes in overall active ensemble size (measured over an entire imaging session) and Ca3. Similarly, when only "total locomotion" is presented as the outcome measure, sub analyses should be performed on richer features of the movement data and presented/discussed to ensure that differential findings are not equally or better explained by differences in forward, reverse, or stalled locomotion or velocity.We agree with the Reviewer that studying how neural activity in motor-related brain regions contributes to movement benefits from drawing comparisons with many behavioral parameters. But our study does not aim to explain what individual SPNs or groups of SPNs encode, or how they affect behavior. Instead, we sought to reveal how dopamine modulates striatal activity, while keeping as many experimental variables constant, including behavior. For this reason, we opted for a single, low-dimensional behavior (forward locomotion), which prior work shows is one of many ‘behavioral syllables’ spontaneously displayed by mice that is associated with the activation of a specific group of SPNs .differential effects on dSPN and iSPN ensembles despite variable effects on behavior strongly suggests that the cellular phenotypes we uncover here reflect the actions of dopamine, as opposed to being indirectly inherited from behavior.We do, however, firmly believe that the reported changes in ensemble size do not stem from differences in velocity or forward, reverse, or stalled locomotion. First, as mentioned above, mice in our assay do not engage in rearward locomotion. Second, our manipulations did not shorten locomotor bout duration, indicating that stalled locomotion is unlikely to contribute to the observed effects on SPN ensemble size. Third, behavioral variables were not consistently affected by pharmacological manipulations . Fourth, we controlled for differences in locomotion frequency, duration and velocity by performing  or repeating  manipulations on motorized treadmills imposing a fixed number of locomotor bouts at set velocities. Fifth, dopamine-mediated changes in the percentage of imaged SPNs that are active during spontaneous locomotion were observed at all recorded velocities . Lastly, the fact that diverse manipulations  produced consistent, Reviewer #2 (Recommendations for the authors):1. While D1 receptors, at least within the striatum, are relatively selective in their expression on dSPNs, D2 antagonists or agonists may act on iSPNs themselves, or local microcircuit elements, including cholinergic interneurons, dopamine terminals, and potentially local glutamatergic and GABAergic synapses. The authors allude to the glutamatergic inputs in the discussion, but one question in my mind was how much of the results here could be related to D2 autoreceptors on dopamine terminals. For example, D2 antagonists presynaptically increase striatal dopamine release, and D2 agonists decrease dopamine release . At the very least, it would be worth discussing the potential mechanisms of the pharmacology.The Reviewer is correct that dopamine receptors localize to many cells and processes in striatum in addition to dSPNs and iSPNs, making it difficult to attribute the effects of dopamine receptor agonists and antagonists to any one element. We first wish to clarify that the intent of this study is to reveal the net effect of dopamine modulation on striatal output in vivo, not to uncover the precise circuit elements or specific receptor types through which it does so. For this reason, we broadly stimulated or blocked all dopamine receptors simultaneously using cocktails of high-affinity D1/2R agonists or antagonists, respectively. One advantage of this approach is that we can a priori exclude confounds arising from secondary changes in presynaptic release of dopamine, since dopamine receptors are already pharmacologically blocked or stimulated. For instance, a presynaptic increase in dopamine release following D2R blockade ought to be of little consequence if all dopamine receptors in striatum are already blocked. Conversely, a decrease in dopamine release following D2R activation may be less impactful on SPNs whose receptors are coincidentally stimulated with D1/2 agonists.Figure 4A) in a small cohort of 3 mice treated with the D1R agonist SKF81297 alone at 3 mg/kg:We nevertheless performed experiments to mitigate concerns that modulation of presynaptic D2Rs significantly contributes to the observed effects. First, we replicated the contraction of dSPN ensembles observed with high dose D1/2R agonists  were undistinguishable from those observed 24 h following the loss of SNc neurons with 6-OHDA, suggesting that presynaptic modulation of DA release via D2 autoreceptors does not significantly contribute to the observed changes in SPN ensemble size.2. Related to the above comment, though some of the differences between healthy and 6-OHDA treated animals in their response to dopamine drugs may be related to chronic changes in synaptic connectivity or intrinsic properties, some may relate to the loss of one of the targets of these drugs: the dopamine terminals. Might the effects of D2 antagonists in healthy animals be an interplay between direct effects on SPNs themselves, plus changes in dopamine release mediated by actions on dopamine terminals? And one of the reasons it's so different in 6-OHDA treated animals is that now this element has been removed?As discussed above, despite the strong influence of D2 autoreceptors on dopamine release from SNc axons, we do not have evidence that this modulation contributes to the effects of D1/2R antagonists or 6-OHDA lesions on the size of dSPN ensembles.3. The authors refer to L-DOPA-induced dyskinesia in their discussion, and compare their findings to published work with L-DOPA-induced dyskinesia, but this behavioral phenomenon was not mentioned/quantified in the text. Based on my understanding of the literature, the severity of dopamine depletion they induced  combined with a dose of 10 mg/kg of L-DOPA would likely induce dyskinesia . Did their animals show dyskinesia? And if so, how did this relate to the patterns of neural activity reported?We unfortunately did not perform detailed behavioral analyses using the validated Abnormal Involuntary Movement (AIM) scale. Axial, forelimb and orolingual AIMs typically develop in mice with unilateral dopamine neuron lesions upon chronic treatment with levodopa . We selected an intermediate dose and treated our mice only once, as we wished to restore striatal dopamine while avoiding severe dyskinetic episodes that may prevent mice from locomoting on the treadmill. We imaged striatal activity prior to levodopa, then administered levodopa before monitoring turning bias in a closed-arm plus-maze to confirm levodopa’s effectiveness, and finally imaged striatal activity again. The strong contraversive turning bias observed  is reminiscent of axial AIMs. However, we did not notice forelimb or orolingual AIMs, suggesting that our mice are unlikely to qualify as dyskinetic according to established standards. In agreement with this, our mice were able to locomote on the treadmill while head-fixed without apparent difficulty. That said, we agree with the Reviewer that it would be interesting for future investigations to relate SPN ensemble size to AIM scores to determine whether the dramatic expansion in dSPN ensemble size predisposes, or directly contributes to dyskinesia.4. For interpretability and clarity, it would be helpful to include details of some major methods in the Results text and/or main Figure legends. For example, the authors should indicate in the Results and/or Figure legends that they used both an A2a-Cre x Ai14 cross and a viral approach to directly label iSPNs, that they used a cocktail of SCH23390 and raclopride to block D1/2 type dopamine receptors, or a cocktail of SKF81297 and quinpirole to activate D1/2 type dopamine receptors.As suggested, the revised version of the manuscript specifies in the Results section that we used transgenic (A2a-Cre::Ai14 mice) as well as viral approaches (A2A-Cre with Cre-dependent tdTomato AAV) to label iSPNs in striatum (lines 125-129). The specific D1 and D2 receptor agonists and antagonists administered are now also indicated in the Results session (lines 191 and 222 in the revised manuscript).5. It is unclear between the Figure and methods text what concentration of L-DOPA was used – the figure itself has "10" listed underneath , but the methods list 12 mg/kg (which may instead refer to benserazide dosing?).We apologize for this typo. The figure reflected the correct L-DOPA dose (10 mg/kg), while the Methods section erroneously listed 12 mg/kg, which the Reviewer rightly guessed referred to benserazide dosage. This error has now been corrected.


Although antimicrobial consumption is considered the main driver of antimicrobial resistance (AMR), other factors probably have a significant but less studied impact. The study’s goal was to assess AMR drivers across different European countries and quantify their possible contributions using the latest data available. Using the ESAC-Net  database, the ESVAC  database and the OECD (Organisation for Economic Cooperation and Development) information, a dataset including 23 European countries was created. Associations between AMR and potential contributing factors were assessed using bivariate correlation and multiple linear regression models for multivariable analyses. Factors associated with the AMR rate among European countries were human ambulatory consumption of antibiotics and per capita expenditure on health, meaning that the higher human ambulatory consumption of antibiotics and the lower the per capita expenditure on health, the higher the AMR. Both variables together explain 74% of AMR variation. Private expenditure on health in terms of % GDP (Gross Domestic Profit) was positively related to a higher AMR rate. In conclusion, considering antibiotic consumption as the most important factor contributing to AMR may be a deviant focus, as resistance transmission may be paramount for AMR levels. Low per capita expenditure on health, probably a surrogate of worse healthcare conditions and a high level of resistance transmission, has a strong correlation with the AMR rate. Increasing public expenditure on healthcare, to strengthen infection control structures and processes interventions, seems relevant to tackle antimicrobial resistance at the European scale. K. pneumoniae resistant to carbapenems increased six-fold. The number of deaths attributable to infections due to third-generation cephalosporin-resistant E. coli increased four-fold [Antimicrobial resistance (AMR) is a significant cause of morbidity and mortality globally ,2,3. Mulour-fold . Currentour-fold .Antibiotic consumption is considered the main force driving the increase in AMR . The assHowever, other factors may contribute to an increased prevalence of AMR. In fact, although antibiotic usage is an important driver for increasing antimicrobial resistance levels, the spreads of resistant bacteria or the genes that encode resistance are likely to be at least as important in the dissemination and prevalence of antimicrobial resistance . The quaThere are a few studies that assess the impact of socioeconomic conditions and health systems organization on AMR, at the same time they assess human and animal consumption, so it makes sense to conduct this study aiming to assess the relative weight of different variables on explaining the heterogeneity of AMR among European countries.The European countries included in our research were: Austria, Belgium, Czech Republic, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, The Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain and the United Kingdom.Using three sources of data  we created a dataset for 23 European countries.Staphylococcus aureus resistant to methicillin (MRSA), Enterococcus faecium and Enterococcus faecalis resistant to vancomycin, Enterobacteriaceae resistant to third-generation cephalosporins, and Pseudomonas aeruginosa and Acinetobacter baumannii resistant to carbapenems. The percentage of resistant isolates was not calculated when less than 10 isolates, with known AST results, were reported. The composite AMR index at the country level was validated by examining the correlation with the composite AMR index calculated from 2016 EARS-Net data. This includes all index components except for AST results for Enterobacterales other than Escherichia coli and Klebsiella pneumonia, because they are not included in EARS-Net.Data for antimicrobial resistance and healthcare-associated infections were obtained from the Health at a Glance 2019  report, https://www.ecdc.europa.eu/en/antimicrobial-consumption/database/rates-country, accessed on 26 June 2020).Data for human antimicrobial consumption for 2016 was retrieved from the European Surveillance of Antimicrobial Consumption Network (ESAC-Net) database. ESAC-Net (formerly ESAC) is a Europe-wide network of national surveillance systems, providing European reference data on antimicrobial consumption in the human sector. It collects and analyses data on human antimicrobial consumption, both in the community and hospital sector. Data considered refers to antibacterials for systemic use (ATC group J01). .Data for veterinary antimicrobial consumption for 2016 was retrieved from the European Surveillance of Veterinary Antimicrobial Consumption (ESVAC) database. ESVAC is a Europe-wide network of national surveillance systems, providing European reference data on antimicrobial consumption in the veterinary sector. .Data for expenditure on health and years lost for 2016 was retrieved from the Organisation for Economic Cooperation and Development (OECD) website. The OECD is an international organisation that works to build better policies and that collects, analyses and shares data from all its participating countries. Data refers to expenditure on health , per capita, current prices in Euro , share of gross domestic product; Government/compulsory schemes expenditure on health , share of gross domestic product; and years lost, /100,000 population, aged 75 years old. , and the second one using a stepwise adjustment of the remaining variables.Twenty-three countries were considered, including 21 EU Member States and two EEA countries (Iceland and Norway). With at least one healthcare-associated infection, the average percentage of hospitalized patients was 4.96%, varying between 2.95% and 8.10%. The average proportion of bacteria resistant to antibiotics, isolated from these infections, was 29.9%, with a country range of 10.69% to 68.42%. The statistical results for all the variables included in the study, for the 23 countries, in 2016 are presented in Overall bivariate correlation analysis of the several variables with the AMR rate across the European countries reveals that there is a closer, and often significant, correlation with the variables related to expenditure on health than with human or veterinary consumption of antimicrobials .Some non-parametric coefficients (Spearman’s rho) tend to be higher than their respective parametric counterpart (Person’s R). This difference hints at potential non-linear relations of these variables with the AMR rate across European countries.p = 0.006).After adjustment for expenditure on health per capita, human antimicrobials consumption becomes partially highly correlated with the rate of AMR, and this partial correlation is higher for the human ambulatory consumption  and Finland (better than expected). Graphic visualisation available at A direct relation of AMR rate with years of life lost seems to depart from linearity. However, it does not reach statistical evidence, only a visual hint. Graphic visualisation available at The results of the multiple linear regression confirm that health expenditure, per capita and human ambulatory antimicrobial consumption, are inversely and directly related to the AMR rate, respectively .The relation of AMR rate and expenditure on health per capita seemed to be slightly non-linear a, so a tOur study shows that using a simple stepwise multiple regression analysis, the factors associated with the rate of AMR among European countries are: human ambulatory consumption of antibiotics; and per capita expenditure on health. Meaning that the higher human ambulatory consumption of antibiotics and the lower the per capita expenditure on health are, the higher the AMR is. Both variables together explain 74% of AMR variation. Additionally, the higher the percentage of private health expenditure among global health expenditures is, the higher the rate of antimicrobial resistance is.This research adds to very few others that emphasise that AMR determinants go far beyond antimicrobial consumption and include socioeconomic and health organisation factors.The data hints that the relation between the AMR rate and expenditure on health per capita may be non-linear. A more complex regression model was obtained with a potential explanation of 94% of the AMR rate variation across countries. This model, essentially, points to the same relations seen in the simpler model. When accounting for the nonlinearity of the expenditure per capita, the AMR rate reveals an additional monetary investment relation, bringing to the equation the private expenditure on health (positively relating it to AMR) while maintaining some important relation with global ambulatory antimicrobials consumption. This model must carefully be interpreted due to the sample size analysed; nevertheless, it offers itself up to interesting interpretation.Enterobacteriaceae [The association between antimicrobial consumption and AMR was demonstrated in several studies, for different pathogens and antimicrobials and in different settings—hospitals, communities, primary care centres, and countries. This kind of context was also reiterated in a recent point prevalence survey in European acute care hospitals, using a composite index of AMR and the percentage of carbapenem-resistant eriaceae . Converseriaceae  found theriaceae .The socio-economic development status of a country understandably influences the success of infection control policies. Per capita expenditure on health was also associated with AMR in a recent study carried worldwide . We may One of our study limitations is the fact that it uses a specific moment of a specific year and that hospital infection and antimicrobial resistance rates are prevalence and not incidence rates. However, resistance data obtained from the Point Prevalence Study largely reflects incidence data obtained in the EARS-Net .The same temporal scale was considered for all the countries and variables in this study . Regarding spatial scale, and considering the European region studied as one spatial region, the aggregation bias may be considered diluted as data collection included 23 data collection location points (countries).Regarding socio-economic data of 2016, it does not significantly deviate from data collected for previous years in each country.We were not able to find a relation between veterinary antimicrobial consumption and AMR rate. According to ESVAC report for 2016 , variatiNo database was found for agriculture consumption of antibiotics or for resistant bacteria in animals and vegetables at the European Union level. However, a recent publication shows the presence of the plasmid-mediated colistin resistance (PMCR)—encoding gene mcr-1—in an Escherichia coli isolate, recovered from lettuce produced and marketed in Portugal . ColistiOur study shows that higher human ambulatory consumption of antibiotics, lower per capita expenditure on health, and a higher percentage of private health expenditure are associated with a higher rate of antimicrobial resistance. Low per capita expenditure on health may be a surrogate of worse healthcare conditions—infrastructure, human resources, and organization. A high percentage of private health expenditure may be a surrogate of less regulated antimicrobial prescriptions.Our findings may have major policy implications. It may be that a quasi-total focus put on reducing human and animal antibiotic consumption as the most important factors contributing to antimicrobial resistance may be a deviant focus, as resistance transmission may be paramount for antimicrobial resistance levels. Therefore, increasing public expenditure on healthcare, promoting interventions to improve sanitation, infection control and prevention, adequate human resources, better infrastructure, and One Health governance are jointly necessary to tackle antimicrobial resistance at the European scale.