Reward expectation enhances action-related activity of nigral dopaminergic and two striatal output pathways.

Neurons comprising nigrostriatal system play important roles in action selection. However, it remains unclear how this system integrates recent outcome information with current action (movement) and outcome (reward or no reward) information to achieve appropriate subsequent action. We examined how neuronal activity of substantia nigra pars compacta (SNc) and dorsal striatum reflects the level of reward expectation from recent outcomes in rats performing a reward-based choice task. Movement-related activity of direct and indirect pathway striatal projection neurons (dSPNs and iSPNs, respectively) were enhanced by reward expectation, similarly to the SNc dopaminergic neurons, in both medial and lateral nigrostriatal projections. Given the classical basal ganglia model wherein dopamine stimulates dSPNs and suppresses iSPNs through distinct dopamine receptors, dopamine might not be the primary driver of iSPN activity increasing following higher reward expectation. In contrast, outcome-related activity was affected by reward expectation in line with the classical model and reinforcement learning theory, suggesting purposive effects of reward expectation.

Potential UV-Protective Effect of Freestanding Biodegradable Nanosheet-Based Sunscreen Preparations in XPA-Deficient Mice.

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disorder. As patients with XP are deficient in nucleotide excision repair, they show severe photosensitivity symptoms. Although skin protection from ultraviolet (UV) radiation is essential to improve the life expectancy of such patients, the optimal protective effect is not achieved even with sunscreen application, owing to the low usability of the preparations. Nanosheets are two-dimensional nanostructures with a thickness in the nanometer range. The extremely large aspect ratios of the nanosheets result in high transparency, flexibility, and adhesiveness. Moreover, their high moisture permeability enables their application to any area of the skin for a long time. We fabricated preparations containing avobenzone (BMDBM) based on freestanding poly (L-lactic acid) (PLLA) nanosheets through a spin-coating process. Although monolayered PLLA nanosheets did not contain enough BMDBM to protect against UV radiation, the layered nanosheets, consisting of five discrete BMDBM nanosheets, showed high UV absorbance without lowering the adhesive strength against skin. Inflammatory reactions in XPA-deficient mice after UV radiation were completely suppressed by the application of BMDBM-layered nanosheets to the skin. Thus, the BMDBM layered nanosheet could serve as a potential sunscreen preparation to improve the quality of life of patients with XP.

Somatic mutations in oral squamous cell carcinomas in 98 Japanese patients and their clinical implications.

INTRODUCTION: The somatic mutational profile of oral squamous cell carcinoma (OSCC) among Japanese patients has been less investigated, partly because of the rarity of the tumor. Moreover, previous studies have either used formalin-fixed paraffin-embedded samples or lacked paired normal tissues. We aimed to determine somatic mutations in the exomes of 76 genes, including 50 driver genes of solid cancers and NOTCH-related genes, some of which are previously reported as frequently mutated in head and neck squamous cell carcinoma or OSCC. MATERIALS AND METHODS: We used fresh-frozen tumor/normal-paired samples from 98 treatment-naïve Japanese patients with OSCC and analyzed their correlations with clinicopathological characteristics and survival. RESULTS: We identified 136 exonic mutations, including 78 non-synonymous mutations, 13 synonymous mutations, 22 nonsense mutations, 2 non-frameshift deletions, 11 frameshift deletion, and 5 each of splice-site and frameshift insertions. The most frequently mutated genes were TP53 (36.7%), FAT1 (9.2%), NOTCH1 (8.2%), CDKN2A (7.1%), ZFHX4 (5.1%), CASP8 (4.1%), EP300 (4.1%), and KMT2D (4.1%). We followed up 90 of the 98 patients for 3 years. Among them, TP53 mutation was associated with significantly shorter 3-year disease-free survival. Most of the identified TP53 mutations occurred in the DNA-binding domain and were functionally deleterious. DISCUSSION: Our findings and the mutation spectra can contribute to the development of a therapeutic strategy for Japanese patients with OSCC.

Deletion of lncRNA XACT does not change expression dosage of X-linked genes, but affects differentiation potential in hPSCs.

Female human pluripotent stem cells (hPSCs) regularly show erosion of X chromosome inactivation featured by the loss of the long non-coding (lnc) RNA XIST and the accumulation of lncXACT. Here, we report that a common mechanism for the initiation of erosion depends on XIST loss but not XACT accumulation on inactive X chromosomes. We further demonstrate that XACT deletion does not affect X-linked gene dosage in eroded hPSCs and that aberrant XIST RNA diffusion induced by the CRISPR activation system is independent of the presence of XACT RNA. In contrast, the deletion of XACT results in the upregulation of neuron-related genes, facilitating neural differentiation in both male and eroded female hPSCs. XACT RNA repression by CRIPSR inhibition results in the same phenotype. Our study finds that XACT is dispensable for maintaining the erosion of X-lined gene repression on inactive X chromosomes but affects neural differentiation in hPSCs.

AMBRA1 controls antigen-driven activation and proliferation of naive T cells.

AMBRA1 (activating molecule in Beclin1-regulated autophagy) is a member of the BECN1 (BECLIN1) protein complex, and it plays a role in autophagy, cell death, tumorigenesis and proliferation. We recently reported that on T-cell receptor (TCR) stimulation, AMBRA1 controlled both autophagy and the cell cycle with metabolic regulation. Accumulating evidence has shown that autophagy and metabolic control are pivotal for T-cell activation, clonal expansion and effector/memory cell fate decision. However, it is unknown whether AMBRA1 is involved in T-cell function under physiological conditions. We found that T cells in Ambra1-conditional knockout (cKO) mice induced an exacerbated graft versus host response when they were transplanted into allogeneic BALB/c mice. Furthermore, Ambra1-deficient T cells showed increased proliferation and cytotoxic capability toward specific antigens in response to in vivo stimulation using allogeneic spleen cells. This enhanced immune response mainly contributed to naive T-cell hyperactivity. The T-cell hyperactivity observed in this study was similar to those in some metabolic factor-deficient mice, but not those in other pro-autophagic factor-deficient mice. Under the static condition, however, naive T cells were reduced in Ambra1-cKO mice, the same as in pro-autophagic factor-deficient mice. Collectively, these results suggested that AMBRA1 was involved in regulating T cell-mediated immune responses through autophagy-dependent and -independent mechanisms.

Tonically Active Neurons in the Monkey Dorsal Striatum Signal Outcome Feedback during Trial-and-error Search Behavior.

An animal's choice behavior is shaped by the outcome feedback from selected actions in a trial-and-error approach. Tonically active neurons (TANs), presumed cholinergic interneurons in the striatum, are thought to be involved in the learning and performance of reward-directed behaviors, but it remains unclear how TANs are involved in shaping reward-directed choice behaviors based on the outcome feedback. To this end, we recorded activity of TANs from the dorsal striatum of two macaque monkeys (Macaca fuscata; 1 male, 1 female) while they performed a multi-step choice task to obtain multiple rewards. In this task, the monkeys first searched for a rewarding target from among three alternatives in a trial-and-error manner and then earned additional rewards by repeatedly choosing the rewarded target. We found that a considerable proportion of TANs selectively responded to either the reward or the no-reward outcome feedback during the trial-and-error search, but these feedback responses were not observed during repeat trials. Moreover, the feedback responses of TANs were similarly observed in any search trials, without distinctions regarding the predicted probability of rewards and the location of chosen targets. Unambiguously, TANs detected reward and no-reward feedback specifically when the monkeys performed trial-and-error searches, in which the monkeys were learning the value of the targets and adjusting their subsequent choice behavior based on the reward and no-reward feedback. These results suggest that striatal cholinergic interneurons signal outcome feedback specifically during search behavior, in circumstances where the choice outcomes cannot be predicted with certainty by the animals.

Alopecia areata susceptibility variant in MHC region impacts expressions of genes contributing to hair keratinization and is involved in hair loss.

BACKGROUND: Alopecia areata (AA) is considered a highly heritable, T-cell-mediated autoimmune disease of the hair follicle. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a genome region known to be associated with AA as compared to other regions. METHODS: We engineered mice carrying AA risk allele identified by haplotype sequencing for the MHC region using allele-specific genome editing with the CRISPR/Cas9 system. Finally, we performed functional evaluations in the mice and AA patients with and without the risk allele. FINDINGS: We identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Furthermore, mice engineered to carry the risk allele displayed a hair loss phenotype. Transcriptomics further identified CCHCR1 as a novel component interacting with hair cortex keratin in hair shafts. Both, these alopecic mice and AA patients with the risk allele displayed morphologically impaired hair and comparable differential expression of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). INTERPRETATION: Our results implicate CCHCR1 with the risk allele in a previously unidentified subtype of AA based on aberrant keratinization in addition to autoimmune events. FUNDING: This work was supported by JSPS KAKENHI (JP16K10177) and the NIHR UCLH Biomedical Research center (BRC84/CN/SB/5984).

Topographic distinction in long-term value signals between presumed dopamine neurons and presumed striatal projection neurons in behaving monkeys.

Nigrostriatal dopamine (DA) projections are anatomically organized along the dorsolateral-ventromedial axis, conveying long-term value signals to the striatum for shaping actions toward multiple future rewards. The present study examines whether the topographic organization of long-term value signals are observed upon activity of presumed DA neurons and presumed striatal projection neurons (phasically active neurons, PANs), as predicted based on anatomical literature. Our results indicate that DA neurons in the dorsolateral midbrain encode long-term value signals on a short timescale, while ventromedial midbrain DA neurons encode such signals on a relatively longer timescale. Activity of the PANs in the dorsal striatum is more heterogeneous for encoding long-term values, although significant differences in long-term value signals were observed between the caudate nucleus and putamen. These findings suggest that topographic DA signals for long-term values are not simply transferred to striatal neurons, possibly due to the contribution of other projections to the striatum.

Toward global standardization of conducting fair investigations of allegations of research misconduct.

In the United States, through nation-wide discussions, the procedures for handling allegations of research misconduct are now well established. Procedures are geared toward carefully treating both complainants and respondents fairly in accordance with the US framework. Other countries, which have their own cultural and legal framework, also need fair and legally compatible procedures for conducting investigations of allegations of research misconduct. Given the rapid growth of international collaboration in research, it is desirable to have a global standard, or common ground, for misconduct investigations. Institutions need clear guidance on important subjects such as what information should be included in the investigation reports, how the investigation committee should be organized once research misconduct allegation has been received, how to conduct the investigation, how the data and information obtained should be taken as evidence for vs. against misconduct, and what policies the investigation committee should follow. We explore these issues from the viewpoint of members of committees investigating accusations of research misconduct (hereafter referred to as "investigation committees") as well as persons overseeing the committees in Japan. We hope to engender productive discussions among experts in misconduct investigations, leading to a formulation of international standards for such investigation.

Imprinted X-chromosome inactivation impacts primitive endoderm differentiation in mouse blastocysts.

Epigenetic and transcriptome alterations are essential for lineage specification, represented by imprinted X-chromosome inactivation (iXCI) in female mouse preimplantation embryos. However, how various factors affect transcriptome states and lineage commitment remains unclear. We found that in vitro culture duration strongly influences transcriptional variation compared to iXCI loss. Single-cell analysis of the inner cell mass (ICM) for major transcription and epigenomic factors revealed that sex-specific differences in expression are diminished by loss of iXCI in the primitive endoderm (PrE) but not in the epiblast. Females had a higher proportion of ICM compared to that in males, and PrE development was affected by iXCI states in female embryos. Our findings provide insight into sex differences and iXCI function in lineage specification.

New PCNT candidate missense variant in a patient with oral and maxillofacial osteodysplasia: a case report.

BACKGROUND: Osteodysplasia of the oral and maxillofacial bone is generally accompanied by systemic bone abnormalities (such as short stature, joint contracture) or other systemic abnormalities (such as renal, dermatological, cardiovascular, optic, or hearing disorders). However, it does not always present this way. Recent reports have suggested that genome-wide sequencing is an effective method for identifying rare or new disorders. Here, we performed whole-exome sequencing (WES) in a patient with a unique form of acquired, local osteodysplasia of the oral and maxillofacial region. CASE PRESENTATION: A 46-year-old woman presented to our hospital with the complaint of gradually moving mandibular teeth (for 6 months), changing facial appearance, and acquired osteolysis of the oral and maxillofacial bones, showing mandibular hypoplasia without family history. Upon skeletal examination, there were no abnormal findings outside of the oral and maxillofacial area; the patient had a height of 157 cm and bone mineral density (according to dual energy x-ray absorptiometry) of 90%. Results of blood and urine tests, including evaluation of bone metabolism markers and neurological and cardiovascular examinations, were normal. We performed WES of genomic DNA extracted from the blood of this patient and her mother, who did not have the disease, as a negative control. We identified 83 new missense variants in the patient, not detected in her mother, including a candidate single nucleotide variant in exon 14 of PCNT (pericentrin). Critical homozygous or compound heterozygous variants in PCNT are a known cause of microcephalic osteodysplastic primordial dwarfism type II accompanied by mandibular hypoplasia, which is similar to the maxillofacial phenotype in this patient. CONCLUSIONS: Protein simulations performed using Polymorphism Phenotyping v2 and Combined Annotation Dependent Depletion software indicated that this missense variant is likely to disrupt the PCNT protein structure. These results suggest that this is a new form of osteolysis related to this PCNT variant.

Interactive effects of OXTR and GAD1 on envy-associated behaviors and neural responses.

Inequity aversion (negative feelings induced by outcome differences between the self and other) plays a key role in human social behaviors. The neurotransmitters oxytocin and GABA have been implicated in neural responses to inequity. However, it remains poorly understood not only how individual genetic factors related to oxytocin and GABA affect the neural mechanisms behind inequity aversion, but also how these genes interact. To address these issues, we examined relationships between genotypes, behavioral decisions and brain activities during the ultimatum game. We identified interactive effects between the polymorphisms of the oxytocin receptor gene (OXTR) and glutamate decarboxylase 1 gene for GABA synthesis (GAD1) on envy aversion (i.e., disadvantageous inequity aversion) and on envy-induced activity in the dorsal ACC (dACC). Thus, our integrated approach suggested interactive genetic effects between OXTR and GAD1 on envy aversion and the underlying neural substrates.

Area-specific Modulation of Functional Cortical Activity During Block-based and Trial-based Proactive Inhibition.

Animals can suppress their behavioral response in advance according to changes in environmental context (proactive inhibition: delaying the start of response), a process in which several cortical areas may participate. However, it remains unclear how this process is adaptively regulated according to contextual changes on different timescales. To address the issue, we used an improved stop-signal task paradigm to behaviorally and electrophysiologically characterize the temporal aspect of proactive inhibition in head-fixed rats. In the task, they must respond to a go cue as quickly as possible (go trial), but did not have to respond if a stop cue followed the go cue (stop trial). The task alternated between a block of only go trials (G-block) and a block of go-and-stop trials (GS-block). We observed block-based and trial-based proactive inhibition (emerging in GS-block and after stop trial, respectively) by behaviorally evaluating the delay in reaction time in correct go trials depending on contextual changes on different timescales. We electrophysiologically analyzed task-related neuronal activity in the primary and secondary motor, posterior parietal, and orbitofrontal cortices (M1, M2, PPC, and OFC, respectively). Under block-based proactive inhibition, spike activity of cue-preferring OFC neurons was attenuated continuously, while M1 and M2 activity was enhanced during motor preparation. Subsequently, M1 activity was attenuated during motor decision/execution. Under trial-based proactive inhibition, the OFC activity was continuously enhanced, and PPC and M1 activity was also enhanced shortly during motor decision/execution. These results suggest that different cortical mechanisms underlie the two types of proactive inhibition in rodents.

Monitoring and Updating of Action Selection for Goal-Directed Behavior through the Striatal Direct and Indirect Pathways.

The basal ganglia play key roles in adaptive behaviors guided by reward and punishment. However, despite accumulating knowledge, few studies have tested how heterogeneous signals in the basal ganglia are organized and coordinated for goal-directed behavior. In this study, we investigated neuronal signals of the direct and indirect pathways of the basal ganglia as rats performed a lever push/pull task for a probabilistic reward. In the dorsomedial striatum, we found that optogenetically and electrophysiologically identified direct pathway neurons encoded reward outcomes, whereas indirect pathway neurons encoded no-reward outcome and next-action selection. Outcome coding occurred in association with the chosen action. In support of pathway-specific neuronal coding, light activation induced a bias on repeat selection of the same action in the direct pathway, but on switch selection in the indirect pathway. Our data reveal the mechanisms underlying monitoring and updating of action selection for goal-directed behavior through basal ganglia circuits.

Roles of centromedian parafascicular nuclei of thalamus and cholinergic interneurons in the dorsal striatum in associative learning of environmental events.

The thalamus provides a massive input to the striatum, but despite accumulating evidence, the functions of this system remain unclear. It is known, however, that the centromedian (CM) and parafascicular (Pf) nuclei of the thalamus can strongly influence particular striatal neuron subtypes, notably including the cholinergic interneurons of the striatum (CINs), key regulators of striatal function. Here, we highlight the thalamostriatal system through the CM-Pf to striatal CINs. We consider how, by virtue of the direct synaptic connections of the CM and PF, their neural activity contributes to the activity of CINs and striatal projection neurons (SPNs). CM-Pf neurons are strongly activated at sudden changes in behavioral context, such as switches in action-outcome contingency or sequence of behavioral requirements, suggesting that their activity may represent change of context operationalized as associability. Striatal CINs, on the other hand, acquire and loose responses to external events associated with particular contexts. In light of this physiological evidence, we propose a hypothesis of the CM-Pf-CINs system, suggesting that it augments associative learning by generating an associability signal and promotes reinforcement learning guided by reward prediction error signals from dopamine-containing neurons. We discuss neuronal circuit and synaptic organizations based on in vivo/in vitro studies that we suppose to underlie our hypothesis. Possible implications of CM-Pf-CINs dysfunction (or degeneration) in brain diseases are also discussed by focusing on Parkinson's disease.

In Vivo Spiking Dynamics of Intra- and Extratelencephalic Projection Neurons in Rat Motor Cortex.

In motor cortex, 2 types of deep layer pyramidal cells send their axons to other areas: intratelencephalic (IT)-type neurons specifically project bilaterally to the cerebral cortex and striatum, whereas neurons of the extratelencephalic (ET)-type, termed conventionally pyramidal tract-type, project ipsilaterally to the thalamus and other areas. Although they have totally different synaptic and membrane potential properties in vitro, little is known about the differences between them in ongoing spiking dynamics in vivo. We identified IT-type and ET-type neurons, as well as fast-spiking-type interneurons, using novel multineuronal analysis based on optogenetically evoked spike collision along their axons in behaving/resting rats expressing channelrhodopsin-2 (Multi-Linc method). We found "postspike suppression" (~100 ms) as a characteristic of ET-type neurons in spike auto-correlograms, and it remained constant independent of behavioral conditions in functionally different ET-type neurons. Postspike suppression followed even solitary spikes, and spike bursts significantly extended its duration. We also observed relatively strong spike synchrony in pairs containing IT-type neurons. Thus, spiking dynamics in IT-type and ET-type neurons may be optimized differently for precise and coordinated motor control.

AMBRA1 is involved in T cell receptor-mediated metabolic reprogramming through an ATG7-independent pathway.

Metabolic reprogramming contributes to dynamic alteration of cell functions and characteristics. In T cells, TCR-mediated signaling evokes metabolic reprogramming and autophagy. AMBRA1 is known to serve in the facilitation of autophagy and quality control of mitochondria, but the role of AMBRA1 in T cell metabolic alteration is unknown. Here, we show that AMBRA1, but not ATG7, plays a role in TCR-mediated control of glycolytic factors and mitochondrial mass, while both AMBRA1 and ATG7 are required for autolysosome formation. Our results suggested that AMBRA1 is a core factor that controls both autophagy and metabolic regulation.

Distinct Functions of the Primate Putamen Direct and Indirect Pathways in Adaptive Outcome-Based Action Selection.

Cortico-basal ganglia circuits are critical regulators of reward-based decision making. Reinforcement learning models posit that action reward value is encoded by the firing activity of striatal medium spiny neurons (MSNs) and updated upon changing reinforcement contingencies by dopamine (DA) signaling to these neurons. However, it remains unclear how the anatomically distinct direct and indirect pathways through the basal ganglia are involved in updating action reward value under changing contingencies. MSNs of the direct pathway predominantly express DA D1 receptors and those of the indirect pathway predominantly D2 receptors, so we tested for distinct functions in behavioral adaptation by injecting D1 and D2 receptor antagonists into the putamen of two macaque monkeys performing a free choice task for probabilistic reward. In this task, monkeys turned a handle toward either a left or right target depending on an asymmetrically assigned probability of large reward. Reward probabilities of left and right targets changed after 30-150 trials, so the monkeys were required to learn the higher-value target choice based on action-outcome history. In the control condition, the monkeys showed stable selection of the higher-value target (that more likely to yield large reward) and kept choosing the higher-value target regardless of less frequent small reward outcomes. The monkeys also made flexible changes of selection away from the high-value target when two or three small reward outcomes occurred randomly in succession. DA D1 antagonist injection significantly increased the probability of the monkey switching to the alternate target in response to successive small reward outcomes. Conversely, D2 antagonist injection significantly decreased the switching probability. These results suggest distinct functions of D1 and D2 receptor-mediated signaling processes in action selection based on action-outcome history, with D1 receptor-mediated signaling promoting the stable choice of higher-value targets and D2 receptor-mediated signaling promoting a switch in action away from small reward outcomes. Therefore, direct and indirect pathways appear to have complementary functions in maintaining optimal goal-directed action selection and updating action value, which are dependent on D1 and D2 DA receptor signaling.

A mutation in NOTCH1 ligand binding region detected in patients with oral squamous cell carcinoma reduces NOTCH1 oncogenic effect.

NOTCH1 is known as an oncogenic or tumor suppressive gene in solid cancer. NOTCH1 mutations in oral squamous cell carcinoma (OSCC) frequently occur near the ligand-binding region. These mutations change the domain structure of this protein and affect the ligand binding activity. When NOTCH1 is activated by ligand binding, NOTCH1 intracellular domain (NICD) is cleaved from the cell membrane. This study investigated the functional change induced by a NOTCH1 mutation detected in OSCC clinical samples using stable transformant analysis. HEK293 cell lines expressing NOTCH1 wild-type (WT cells) or p.A465T NOTCH1 (A465T cells) were established. NOTCH1 expression was analyzed by flow cytometry, western blotting, and immunofluorescence using an anti-human NOTCH1 antibody. mRNA expression levels in WT and A465T cells were determined by quantitative real-time PCR (qPCR). Cell proliferation was analyzed by using cell growth assays and a xenograft tumor assay. Flow cytometry indicated that NOTCH1 expression on the cell membrane was lower in A465T cells than that in WT cells. NOTCH1 and NICD were both detected by western blot in WT and A465T cells. The immunofluorescence signal for NICD was detected in the nucleus of WT cells, while it was localized mainly in the cytoplasm of A465T cells. HES1 and HEY1 mRNA expression levels were lower in A465T than in WT cells. The cell growth of WT cells was significantly higher than that of HEK293 cells (3-fold, P<0.01), while that of A465T cells was significantly lower than that of HEK293 cells (37%, P<0.01). In a xenograft model, the tumor cell implantation rate of WT cells was 80%, while that of A465T cells was 0%. This study indicates that NOTCH1 acts as an oncogene and that the NOTCH1 mutation (p.A465T) in the ligand-binding region causes the loss of tumorigenicity by downregulating the NOTCH1 pathway.

Microneedle pH Sensor: Direct, Label-Free, Real-Time Detection of Cerebrospinal Fluid and Bladder pH.

Acid-base homeostasis (body pH) inside the body is precisely controlled by the kidneys and lungs and buffer systems, such that even a minor pH change could severely affect many organs. Blood and urine pH tests are common in day-to-day clinical trials and require little effort for diagnosis. There is always a great demand for in vivo testing to understand more about body metabolism and to provide effective diagnosis and therapy. In this article, we report the simple fabrication of microneedle-based direct, label-free, and real-time pH sensors. The reference and working electrodes were Ag/AgCl thick films and ZnO thin films on tungsten (W) microneedles, respectively. The morphological and structural characteristics of microneedles were carefully investigated through various analytical methods. The developed sensor exhibited a Nernstian response of -46 mV/pH. Different conditions were used to test the sensor to confirm their accuracy and stability, such as various buffer solutions, with respect to time, and we compared the reading with commercial pH electrodes. Besides that, the fabricated microneedle sensor ability is proven by in vivo testing in mouse cerebrospinal fluid (CSF) and bladders. The pH sensor procedure reported here is totally reversible, and results were reproducible after several rounds of testing.