Optogenetic stimulation of neurons in the anterior cingulate cortex induces changes in intravesical bladder pressure and the micturition reflex.

Lower urinary tract (LUT) function is controlled by the central nervous system, including higher-order cognitive brain regions. The anterior cingulate cortex (ACC) is one of these regions, but the role of its activity in LUT function remains poorly understood. In the present study, we conducted optogenetic experiments to manipulate neural activity in mouse ACC while monitoring bladder pressure to elucidate how the activity of ACC regulates LUT function. Selective optogenetic stimulation of excitatory neurons in ACC induced a sharp increase in bladder pressure, whereas activation of inhibitory neurons in ACC prolonged the interval between bladder contractions. Pharmacological manipulation of ACC also altered bladder contractions, consistent with those observed in optogenetic experiments. Optogenetic mapping of the cortical area responsible for eliciting the increase in bladder pressure revealed that stimulation to ACC showed more potent effects than the neighboring motor cortical areas. These results suggest that ACC plays a crucial role in initiating the bladder pressure change and the micturition reflex. Thus, the balance between excitation and inhibition in ACC may regulate the reflex bidirectionally.

A multicolor suite for deciphering population coding of calcium and cAMP in vivo.

cAMP is a universal second messenger regulated by various upstream pathways including Ca2+ and G-protein-coupled receptors (GPCRs). To decipher in vivo cAMP dynamics, we rationally designed cAMPinG1, a sensitive genetically encoded green cAMP indicator that outperformed its predecessors in both dynamic range and cAMP affinity. Two-photon cAMPinG1 imaging detected cAMP transients in the somata and dendritic spines of neurons in the mouse visual cortex on the order of tens of seconds. In addition, multicolor imaging with a sensitive red Ca2+ indicator RCaMP3 allowed simultaneous measurement of population patterns in Ca2+ and cAMP in hundreds of neurons. We found Ca2+-related cAMP responses that represented specific information, such as direction selectivity in vision and locomotion, as well as GPCR-related cAMP responses. Overall, our multicolor suite will facilitate analysis of the interaction between the Ca2+, GPCR and cAMP signaling at single-cell resolution both in vitro and in vivo.

Effects of vasodilators on beat-to-beat and every fifteen minutes blood pressure variability induced by noradrenaline infusion in rats.

Increased blood pressure variability (BPV) was shown to be associated with cardiovascular morbidities and/or mortalities. There are various types of BPV depending on time intervals of BP measurements, ranging from beat-to-beat to visit-to-visit or year-to-year. We previously found that continuous infusion of noradrenaline (NA) for 14 days increased short-term BPV every 15 min in rats. The aims of this study were to examine (1) whether NA infusion increases very short-term beat-to-beat BPV, (2) the effects of azelnidipine and hydralazine on NA-induced BPV, and (3) whether baroreceptor reflex sensitivity (BRS) is affected by NA or NA plus those vasodilators. Nine-week-old Wistar rats infused subcutaneously with 30 µg/h NA were orally treated with or without 9.7 mg/day azelnidipine or 5.9 mg/day hydralazine over 14 days. BP levels were continuously monitored via abdominal aortic catheter with a telemetry system in an unrestrained condition. Standard deviations (SDs) were used to evaluate beat-to-beat BPV and BPV every 15 min which was obtained by averaging BP levels for 10-s segment at each time point. BRS was determined by a sequence analysis. Continuous NA infusion over 14 days increased average BP, beat-to-beat BPV, and BPV every 15 min, lowering BRS. Comparing the two vasodilators, hydralazine reduced BP elevation by NA; meanwhile, azelnidipine alleviated BPV augmentation, preserving BRS, despite a smaller BP reduction. Thus, NA infusion increased both very short- and short-term BPV concomitantly with impaired BRS, while azelnidipine had an inhibitory effect, possibly independent of BP-lowering, on those types of BPV and impairment of BRS.

A multicenter, single-arm, phase II clinical trial of adrenomedullin in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

Background: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary cerebral small vessel disease (SVD), currently lacks disease-modifying treatments. Adrenomedullin (AM), a vasoactive peptide with angiogenic, vasodilatory, anti-inflammatory, and anti-oxidative properties, shows potential effects on the neuro-glial-vascular unit. Objective: The AdrenoMedullin for CADASIL (AMCAD) study aims to assess the efficacy and safety of AM in patients with CADASIL. Sample size: Overall, 60 patients will be recruited. Methods: The AMCAD is a multicenter, investigator-initiated, single-arm phase II trial. Patients with a confirmed CADASIL diagnosis, based on NOTCH3 genetic testing, will receive an 8-h AM treatment (15 ng/kg/min) for 14 days following a baseline assessment (from day 1 to day 14). Follow-up evaluations will be performed on days 15, 28, 90, and 180. Study outcomes: The primary endpoint is the cerebral blood flow change rate in the frontal cortex, evaluated using arterial spin labeling magnetic resonance imaging, from baseline to day 28. Summary statistics, 95% confidence intervals, and a one-sample t-test will be used for analysis. Conclusion: The AMCAD study aims to represent the therapeutic potential of AM in patients with CADASIL, addressing an unmet medical need in this challenging condition. Clinical Trial Registration: jRCT 2,051,210,117 (https://jrct.niph.go.jp/en-latest-detail/jRCT2051210117).

Cerebellar climbing fibers multiplex movement and reward signals during a voluntary movement task in mice.

Cerebellar climbing fibers convey sensorimotor information and their errors, which are used for motor control and learning. Furthermore, they represent reward-related information. Despite such functional diversity of climbing fiber signals, it is still unclear whether each climbing fiber conveys the information of single or multiple modalities and how the climbing fibers conveying different information are distributed over the cerebellar cortex. Here we perform two-photon calcium imaging from cerebellar Purkinje cells in mice engaged in a voluntary forelimb lever-pull task and demonstrate that climbing fiber responses in 68% of Purkinje cells can be explained by the combination of multiple behavioral variables such as lever movement, licking, and reward delivery. Neighboring Purkinje cells exhibit similar climbing fiber response properties, form functional clusters, and share noise fluctuations of responses. Taken together, individual climbing fibers convey behavioral information on multiplex variables and are spatially organized into the functional modules of the cerebellar cortex.

Dynamic organization of cerebellar climbing fiber response and synchrony in multiple functional components reduces dimensions for reinforcement learning.

Cerebellar climbing fibers convey diverse signals, but how they are organized in the compartmental structure of the cerebellar cortex during learning remains largely unclear. We analyzed a large amount of coordinate-localized two-photon imaging data from cerebellar Crus II in mice undergoing 'Go/No-go' reinforcement learning. Tensor component analysis revealed that a majority of climbing fiber inputs to Purkinje cells were reduced to only four functional components, corresponding to accurate timing control of motor initiation related to a Go cue, cognitive error-based learning, reward processing, and inhibition of erroneous behaviors after a No-go cue. Changes in neural activities during learning of the first two components were correlated with corresponding changes in timing control and error learning across animals, indirectly suggesting causal relationships. Spatial distribution of these components coincided well with boundaries of Aldolase-C/zebrin II expression in Purkinje cells, whereas several components are mixed in single neurons. Synchronization within individual components was bidirectionally regulated according to specific task contexts and learning stages. These findings suggest that, in close collaborations with other brain regions including the inferior olive nucleus, the cerebellum, based on anatomical compartments, reduces dimensions of the learning space by dynamically organizing multiple functional components, a feature that may inspire new-generation AI designs.

Natriuretic peptides potentiate cardiac hypertrophic response to noradrenaline in rats.

Excessive activation of the sympathetic nervous system is involved in cardiovascular damage including cardiac hypertrophy. Natriuretic peptides are assumed to exert protective actions for the heart, alleviating hypertrophy and/or fibrosis of the myocardium. In contrast to this assumption, we show in the present study that both atrial and C-type natriuretic peptides (ANP and CNP) potentiate cardiac hypertrophic response to noradrenaline (NA) in rats. Nine-week-old male Wistar rats were continuously infused with subcutaneous 30 micro-g/h NA without or with persistent intravenous administration of either 1.0 micro-g/h ANP or CNP for 14 days. Blood pressure (BP) was recorded under an unrestrained condition by a radiotelemetry system. Cardiac hypertrophic response to NA was evaluated by heart weight/body weight (HW/BW) ratio and microscopic measurement of myocyte size of the left ventricle. Mean BP levels at the light and dark cycles rose by about 20 mmHg following NA infusion for 14 days, with slight increases in HW/BW ratio and ventricular myocyte size. Infusions of ANP and CNP had no significant effects on mean BP in NA-infused rats, while two natriuretic peptides potentiated cardiac hypertrophic response to NA. Cardiac hypertrophy induced by co-administration of NA and ANP was attenuated by treatment with prazosin or atenolol. In summary, both ANP and CNP potentiated cardiac hypertrophic effect of continuously infused NA in rats, suggesting a possible pro-hypertrophic action of natriuretic peptides on the heart.

Adrenomedullin alleviates mucosal injury in experimental colitis and increases claudin-4 expression in the colonic epithelium.

Adrenomedullin (AM) is a peptide with pleiotropic physiological functions that attenuates intestinal mucosal inflammation. However, the mechanism underpinning mucosal protection by AM is not fully understood, and its effect on intestinal epithelial cells remains unclear. Here, we investigated the effects of AM on junctional molecules in primary-cultured murine intestinal epithelial cells and discovered that AM upregulates claudin-4 expression. In a mouse model of dextran sulfate sodium-induced colitis, AM administration also enhanced claudin-4 expression and accelerated mucosal regeneration. Furthermore, AM reversed TNFα-mediated downregulation of claudin-4 and loss of cell-cell adhesion of the HCT116 human intestinal epithelial cell line in vitro. These results indicate that AM may enhance intestinal epithelial integrity by upregulating claudin-4 expression.

Analysis of Mechanisms for Increased Blood Pressure Variability in Rats Continuously Infused with Angiotensin II.

Objective: We reported that rats infused with angiotensin II (Ang II) are not only a model of hypertension but also of augmented 24 h blood pressure variability (BPV). In this study, we examined the mechanisms for Ang II-induced BPV, focusing on BP, heart rate (HR), baroreceptor reflex sensitivity (BRS), and medial area of the aortic arch. Methods: Nine-week-old male Wistar rats were infused with subcutaneous 5.2 µg/kg/h Ang II with or without oral administration with 30 mg/kg/day azelnidipine for 14 days. BP and HR were recorded every 15 min under an unrestrained condition by a radiotelemetry system, while BPV was evaluated by standard deviation of BP. BRS was quantified by a sequence analysis, and medial thickness of the aortic arch was measured by microscopic examination. Results: BPV increased at days 7 and 14 following continuous infusion of Ang II. Before the infusion, a positive correlation was found between BP and HR, but it became negative at day 7 and then weakened or disappeared at day 14. BRS was slightly impaired at day 7 and significantly lowered at day 14, a phenomenon accompanied by thickened medial area of the aortic arch in Ang II-infused rats. Those Ang II-induced alterations were all significantly attenuated by azelnidipine. Conclusions: The present findings suggest sequential changes in the mechanisms behind augmented BPV in rats continuously infused with Ang II over 14 days.

Development of Long-Acting Human Adrenomedullin Fc-Fusion Proteins.

(1) Background: Human adrenomedullin (hAM) is a hypotensive peptide hormone that exerts powerful anti-inflammatory effects. AM also had therapeutic effects in various animal experimental models of disease. However, treatment required continuous administration as the half-life of native AM is short in blood. To resolve this, we developed four human IgG1 and IgG4 Fc-fusion proteins containing full-length hAM or hAM residues 6-52. (2) Methods: We used mammalian cells to produce recombinant Fc-AM derivatives and tested the pharmacokinetics and biological activity of Fc-AM. (3) Results: We developed four Fc-fusion AMs (Fc-AM), which are long-acting AM derivatives in mammalian cells. Fc-AM had a prolonged half-life in blood and retained its ability to bind to the AM1 receptor. Fc-AM (6-52) induced higher cAMP levels for the receptor than Fc-AM. After the administration of IgG1-AM (6-52) or IgG4-AM (6-52) to rats, tissue transfer to the kidney and small intestine was observed. In addition, treatment with IgG4-AM (6-52) inhibited blood pressure increase in spontaneously hypertensive rats. (4) Conclusions: Fc-AM produced from mammalian cells can be easily prepared and might be an effective novel therapeutic agent.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse using a Large Cranial Window.

Wide-field calcium imaging from the mouse's neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

Adrenomedullin for biologic-resistant Crohn's disease: A randomized, double-blind, placebo-controlled phase 2a clinical trial.

BACKGROUND AND AIM: Adrenomedullin is a bioactive peptide with many pleiotropic effects, including mucosal healing and immunomodulation. Adrenomedullin has shown beneficial effects in rodent models of inflammatory bowel disease and, more importantly, in clinical trials including patients with ulcerative colitis. We performed a successive clinical trial to investigate the efficacy and safety of adrenomedullin in patients with Crohn's disease (CD). METHODS: This was a multicenter, double-blind, placebo-controlled phase 2a trial that evaluated 24 patients with biologic-resistant CD in Japan. Patients were randomly assigned to three groups and were given an infusion of 10 or 15 ng/kg/min of adrenomedullin or placebo for 8 h per day for 7 days. The primary endpoint was the change in the CD activity index (CDAI) at 8 weeks. The main secondary endpoints included changes in CDAI from week 4 to week 24. RESULTS: No differences in the primary or secondary endpoints were observed between the three groups by the 8th week. Changes in CDAI in the placebo group gradually decreased and disappeared at 24 weeks, but those in the adrenomedullin-treated groups (10 or 15 ng/kg/min group) remained at steady levels for 24 weeks. Therefore, a significant difference was observed between the placebo and adrenomedullin-treated groups at 24 weeks (P = 0.043) in the mixed-effects model. We noted mild adverse events caused by the vasodilatory effect of adrenomedullin. CONCLUSION: In this trial, we observed a long-lasting (24 weeks) decrease in CDAI in the adrenomedullin-treated groups. Adrenomedullin might be beneficial for biologic-resistant CD, but further research is needed.

A Novel Device of Reaching, Grasping, and Retrieving Task for Head-Fixed Mice.

Reaching, grasping, and retrieving movements are essential to our daily lives and are common in many mammalian species. To understand the mechanism for controlling this movement at the neural circuit level, it is necessary to observe the activity of individual neurons involved in the movement. For stable electrophysiological or optical recordings of neural activity in a behaving animal, head fixation effectively minimizes motion artifacts. Here, we developed a new device that allows mice to perform reaching, grasping, and retrieving movements during head fixation. In this method, agar cubes were presented as target objects in front of water-restricted mice, and the mice were able to reach, grasp, and retrieve them with their forelimb. The agar cubes were supplied by a custom-made automatic dispenser, which uses a microcontroller to control the two motors to push out the agar cubes. This agar presentation system supplied approximately 20 agar cubes in consecutive trials. We confirmed that each agar cube could be presented to the mouse with an average weight of 55 ± 3 mg and positional accuracy of less than 1 mm. Using this system, we showed that head-fixed mice could perform reaching, grasping, and retrieving tasks after 1 week of training. When the agar cube was placed near the mice, they could grasp it with a high success rate without extensive training. On the other hand, when the agar cube was presented far from the mice, the success rate was initially low and increased with subsequent test sessions. Furthermore, we showed that activity in the primary motor cortex is required for reaching movements in this task. Therefore, our system can be used to study neural circuit mechanisms for the control and learning of reaching, grasping, and retrieving movements under head-fixed conditions.

Angiotensin II Induces Aortic Rupture and Dissection in Osteoprotegerin-Deficient Mice.

Background The biological mechanism of action for osteoprotegerin, a soluble decoy receptor for the receptor activator of nuclear factor-kappa B ligand in the vascular structure, has not been elucidated. The study aim was to determine if osteoprotegerin affects aortic structural integrity in angiotensin II (Ang II)-induced hypertension. Methods and Results Mortality was higher (P<0.0001 by log-rank test) in 8-week-old male homozygotes of osteoprotegerin gene-knockout mice given subcutaneous administration of Ang II for 28 days, with an incidence of 21% fatal aortic rupture and 23% aortic dissection, than in age-matched wild-type mice. Ang II-infused aorta of wild-type mice showed that osteoprotegerin immunoreactivity was present with proteoglycan. The absence of osteoprotegerin was associated with decreased medial and adventitial thickness and increased numbers of elastin breaks as well as with increased periostin expression and soluble receptor activator of nuclear factor-kappa B ligand concentrations. PEGylated human recombinant osteoprotegerin administration decreased all-cause mortality (P<0.001 by log-rank test), the incidence of fatal aortic rupture (P=0.08), and aortic dissection (P<0.001) with decreasing numbers of elastin breaks, periostin expressions, and soluble receptor activator of nuclear factor-kappa B ligand concentrations in Ang II-infused osteoprotegerin gene-knockout mice. Conclusions These data suggest that osteoprotegerin protects against aortic rupture and dissection in Ang II-induced hypertension by inhibiting receptor activator of nuclear factor-kappa B ligand activity and periostin expression.

A Flp-dependent G-CaMP9a transgenic mouse for neuronal imaging in vivo.

Genetically encoded calcium indicators (GECIs) are widely used to measure calcium transients in neuronal somata and processes, and their use enables the determination of action potential temporal series in a large population of neurons. Here, we generate a transgenic mouse line expressing a highly sensitive green GECI, G-CaMP9a, in a Flp-dependent manner in excitatory and inhibitory neuronal subpopulations downstream of a strong CAG promoter. Combining this reporter mouse with viral or mouse genetic Flp delivery methods produces a robust and stable G-CaMP9a expression in defined neuronal populations without detectable detrimental effects. In vivo two-photon imaging reveals spontaneous and sensory-evoked calcium transients in excitatory and inhibitory ensembles with cellular resolution. Our results show that this reporter line allows long-term, cell-type-specific investigation of neuronal activity with enhanced resolution in defined populations and facilitates dissecting complex dynamics of neural networks in vivo.

Adrenomedullin Therapy in Moderate to Severe COVID-19.

The 2019 coronavirus (COVID-19) pandemic is still in progress, and a significant number of patients have presented with severe illness. Recently introduced vaccines, antiviral medicines, and antibody formulations can suppress COVID-19 symptoms and decrease the number of patients exhibiting severe disease. However, complete avoidance of severe COVID-19 has not been achieved, and more importantly, there are insufficient methods to treat it. Adrenomedullin (AM) is an endogenous peptide that maintains vascular tone and endothelial barrier function. The AM plasma level is markedly increased during severe inflammatory disorders, such as sepsis, pneumonia, and COVID-19, and is associated with the severity of inflammation and its prognosis. In this study, exogenous AM administration reduced inflammation and related organ damage in rodent models. The results of this study strongly suggest that AM could be an alternative therapy in severe inflammation disorders, including COVID-19. We have previously developed an AM formulation to treat inflammatory bowel disease and are currently conducting an investigator-initiated phase 2a trial for moderate to severe COVID-19 using the same formulation. This review presents the basal AM information and the most recent translational AM/COVID-19 study.

Translational studies of adrenomedullin and related peptides regarding cardiovascular diseases.

Adrenomedullin (AM) is a vasodilative peptide with various physiological functions, including the maintenance of vascular tone and endothelial barrier function. AM levels are markedly increased during severe inflammation, such as that associated with sepsis; thus, AM is expected to be a useful clinical marker and therapeutic agent for inflammation. However, as the increase in AM levels in cardiovascular diseases (CVDs) is relatively low compared to that in infectious diseases, the value of AM as a marker of CVDs seems to be less important. Limitations pertaining to the administrative route and short half-life of AM in the bloodstream (<30 min) restrict the therapeutic applications of AM for CVDs. In early human studies, various applications of AM for CVDs were attempted, including for heart failure, myocardial infarction, pulmonary hypertension, and peripheral artery disease; however, none achieved success. We have developed AM as a therapeutic agent for inflammatory bowel disease in which the vasodilatory effect of AM is minimized. A clinical trial evaluating this AM formulation for acute cerebral infarction is ongoing. We have also developed AM derivatives that exhibit a longer half-life and less vasodilative activity. These AM derivatives can be administered by subcutaneous injection at long-term intervals. Accordingly, these derivatives will reduce the inconvenience in use compared to that for native AM and expand the possible applications of AM for treating CVDs. In this review, we present the latest translational status of AM and its derivatives.

A likely unavoidable clinical scenario during treatment for venous thromboembolism complicated with severe immune thrombocytopenia: A case report.

Patients with immune thrombocytopenia have increased risks of bleeding and thrombosis. The acute-phase treatment for venous thromboembolism complicated with severe immune thrombocytopenia involves a "platelet dilemma" in therapeutic decision-making.

Adrenomedullin: A Novel Therapeutic for the Treatment of Inflammatory Bowel Disease.

Adrenomedullin (AM) is a bioactive peptide with various physiological functions, including vasodilation, angiogenesis, anti-inflammation, organ protection, and tissue repair. AM suppresses inflammatory cytokine production in the intestinal mucosa, improves vascular and lymphatic regeneration and function, mucosal epithelial repair, and immune function in the intestinal bacteria of animal models with intestinal inflammation. We have been promoting translational research to develop novel therapeutic agents for inflammatory bowel disease (IBD) using AM and have started clinical research for IBD patients since 2010. A multicenter clinical trial is currently underway in Japan for patients with refractory ulcerative colitis and Crohn's disease. Moreover, since current AM administration is limited to continuous intravenous infusion, the development of a subcutaneous formulation using long-acting AM is underway for outpatient treatment.

A database and deep learning toolbox for noise-optimized, generalized spike inference from calcium imaging.

Inference of action potentials ('spikes') from neuronal calcium signals is complicated by the scarcity of simultaneous measurements of action potentials and calcium signals ('ground truth'). In this study, we compiled a large, diverse ground truth database from publicly available and newly performed recordings in zebrafish and mice covering a broad range of calcium indicators, cell types and signal-to-noise ratios, comprising a total of more than 35 recording hours from 298 neurons. We developed an algorithm for spike inference (termed CASCADE) that is based on supervised deep networks, takes advantage of the ground truth database, infers absolute spike rates and outperforms existing model-based algorithms. To optimize performance for unseen imaging data, CASCADE retrains itself by resampling ground truth data to match the respective sampling rate and noise level; therefore, no parameters need to be adjusted by the user. In addition, we developed systematic performance assessments for unseen data, openly released a resource toolbox and provide a user-friendly cloud-based implementation.