Mbnl1+/−/Mbnl2−/− mice demonstrate whole-brain volume reductions, ventriculomegaly and regional gray and white matter volume reductions. Comparative MRIs on 2-month-old Mbnl1−/−, Mbnl2−/− and Mbnl1−/−/Mbnl2+/− brains show genotype-specific reductions in white and gray matter volumes. In both cohorts, white matter volume reductions predominate, with Mbnl2 loss leading to more widespread alterations than Mbnl1 loss. Hippocampal volumes are susceptible to changes in either Mbnl1 or Mbnl2 levels, where both single gene and dual depletions result in comparable volume losses. In contrast, the cortex, inter/midbrain, cerebellum and hindbrain regions show both gene and dose-specific volume decreases. Our results provide a molecular explanation for phenotype intensification in congenital DM1 and the variability in the brain structural alterations reported in DM1.Myotonic Dystrophy Type I (DM1) patients demonstrate widespread and variable brain structural alterations whose etiology is unclear. We demonstrate that inactivation of the Muscleblind-like proteins, Mbnl1 and Mbnl2, initiates brain structural defects. 2D FSE T2w MRIs on 4-month-old  Sta Maria et al. use 2D MRI to study brains from mice deficient in Mbnl1 and/or Mbnl2 as these Muscleblind-like proteins are thought to play a role in Myotonic Dystrophy Type I (DM1). They show brain region-specific reductions in white and gray matter that could help to explain the variability in the brain structural alterations reported in DM1. DM1 pathology results primarily from the expression of toxic expanded CUG repeat RNA which dysregulates RNA biology7. RNA processing, localization, and translation defects arise from CUG-expansion RNA-mediated functional alterations of RNA-binding proteins, including the muscleblind-like (MBNL) proteins, CUG-BP1 and hnRNPH15. In addition, CUG-expansion RNAs localize the SHARP transcription factor from the nucleus to the cytoplasm in patient myoblasts to alter steady-state mRNA levels16 and repeat-associated non-AUG (RAN) translation can result in the generation of toxic peptides17. SHARP/Mint inactivation causes severe postnatal brain hypoplasia in mice18 and RAN-translation peptides have been shown to play an important role in the degenerative brain changes observed in ALS and frontotemporal dementia19. The molecular mechanisms underpinning DM1 brain structural alterations are however currently not fully understood.Myotonic dystrophy type I (DM1) is an RNA-dominant multi-system disorder in which the brain is prominently affected4. Genetic anticipation or an increase in symptoms and severity within a pedigree is a consequence of the progressive expansion of the CTG-repeat tract between generations20. Additional sources of phenotypic variability are the stochastic CTG-repeat expansions that occur in somatic cells with time21. Both anticipation and symptom variability are pronounced in the brain, with large, mostly maternally inherited CTG expansions, resulting in severe pathology characterized by mental retardation and autism-like features in the congenital form of the disease20. Smaller expansions inherited from either parent result in milder adult-onset or moderately affected juvenile-onset DM1, where patients can demonstrate mental retardation, deficits in memory and learning, with visuospatial learning and reconstruction being prominently affected, dysexecutive syndrome, and excessive daytime sleepiness. Other behavioral and emotional alterations include anxiety, depression, apathy, attention-deficit hyperactivity disorder, obsessive compulsive disorder, and avoidant personality traits2.DM1 is characterized by genetic anticipation and symptom variability23. As ventricular enlargement, which is documented in greater than 50% of congenital DM1 infants in some studies, is observed at birth, brain structural defects in this group must in part be developmental in origin22. In adult-onset and juvenile-onset DM1, ventricular enlargement is less prominent but can progress with time23. All three DM1 patient classes show regional gray matter atrophy in the cortex, the hippocampus, the basal ganglia and other subcortical regions, and in the cerebellum24. White matter hyperintensities in the external capsule, the cortex, subcortical deep white matter, and in the periventricular regions are observed in all three DM1 classes24. The structure of normal appearing white matter can show widespread impairments, demonstrating both reductions in fractional anisotropy and increased mean diffusivity3. Such alterations are recorded in the corpus callosum, the capsule, and in a majority of the association, projection, and commissural fibers24. Decreases in white matter volumes in the corpus callosum, the fornix, the cingulum bundle, and in the subcortical regions of all lobes are observed in DM1 with the thinning of the corpus callosum being particularly prominent in congenital DM123.In congenital DM1 patients, magnetic resonance imaging (MRI) studies demonstrate microcephaly, global brain atrophy, and prominent ventricular enlargement25. The effect of age and disease-duration studies with matched cohorts further supports progressive gray- and white matter atrophy and increases in ventricular volume with time26. Possible correlations between structural impairments and behavioral alterations in DM1 are unresolved with some studies demonstrating correlative changes in behavior and white matter defects28. Thus, neuroimaging in DM1 patients demonstrates widespread brain involvement and lends support for gray matter atrophy and loss of connectivity between gray matter regions occurring secondarily to white matter breakdown contributing to behavioral changes in DM1 patients28.Longitudinal studies, although rare, demonstrate progressive degenerative changes in white matter, cerebral atrophy, and increases in ventricular volume with time30 support the potential of brain structural defects working in concert with the functional alterations in MBNL1/2-target RNAs in driving behavioral dysfunction in DM1 patients.As one or more molecular defects may work coordinately to impact brain structural integrity in DM1, deciphering the molecular mechanisms that initiate such changes is largely intractable in patients. We therefore carried out MRI studies in mouse models where Mbnl1 and Mbnl2 are inactivated either singly or in composite. This analysis shows widespread gray- and white matter defects resulting from Mbnl1 and Mbnl2 loss and identifies a regulatory role for the Mbnl1 and the Mbnl2 proteins in maintaining the structural integrity of the brain. As the brain structural alterations that manifest with Mbnl1/2 loss recapitulate several prominent DM1 brain structural defects, this study demonstrates that a key mechanism driving the brain structural alterations in DM1 patients is the functional inactivation of the MBNL1 and the MBNL2 proteins by the CUG-expansion RNAs. Correlative changes in hippocampal and white matter volume reductions with the behavioral alterations reported in mouse models depleted of Mbnl1 and Mbnl2Mbnl1+/+/Mbnl2+/+ (n = 5) and 129sv Mbnl1+/−/Mbnl2−/− (n = 5) cohorts as described in the “Methods”. As the Mbnl1−/−/Mbnl2−/− genotype demonstrates an embryonic lethal phenotype and because Mbnl2 levels are higher in the brain when compared with Mbnl1 levels9, Mbnl1+/−/Mbnl2−/− mice should afford the largest Mbnl1/2 reductions in the brain. Four-month-old cohorts were chosen because T2 weighted and diffusion-weighted MRI in mice have been shown that the most dramatic postnatal changes in cortex thickness and myelination are largely over by three months31.To test the hypothesis that Mbnl1 and Mbnl2 regulate the structural integrity of the brain, we developed 4-month-old female (4mo) 129sv Mbnl1+/+/Mbnl2+/+ and Mbnl1+/−/Mbnl2−/− cohorts were subjected to 2D FSE T2w MRI and a montage of extracted whole-brain slices for each subject was assembled with or without overlaid manually drawn, color-filled regions of interest for qualitative assessments. Volumetric measurements of the ROIs were extracted for quantitative analysis as described in the Methods. These montages demonstrated that the total number of slices were reduced by 1–4 slices with regional differences being observed in Mbnl1+/−/Mbnl2−/− brains when compared with Mbnl1+/+/Mbnl2+/+ brains   and the apparent ventricle volume to the whole-brain volume ratio was ~152% increased (p = 0.0001) in Mbnl1+/−/Mbnl2−/− mice when compared with Mbnl1+/+/Mbnl2+/+ controls  Fig. , Table 1ols Fig. , Table 1Mbnl1+/−/Mbnl2−/− mice showed a significant reduction (~21% decrease) in volume (p = 0.0076) when compared with age matched Mbnl1+/+/Mbnl2+/+ mice  at four months (p = 0.0001)  Fig. ; Table 1Mbnl1+/−/Mbnl2−/− mice demonstrated significantly reduced isocortex    but not in the anterior Bregma isocortex, defined as Bregma +   and the hindbrain showing an ~18% decrease (p = 0.0019)  Fig. ; Table 169) Fig. ; Table 1a + Fig. ; Table 164) Fig. ; Table 119) Fig. ; Table 1ice Fig.  Table 1.Mbnl1+/−/Mbnl2−/− mice. It is unclear if this change reflects a bona fide increase or is a compensatory alteration in response to other regional gray- and white matter volume shrinkages.Thus, Mbnl1 heterozygosity, in conjunction with Mbnl2 loss, results in global brain atrophy with increased apparent ventricle volumes and widespread white and gray matter volume reductions in the mouse brain at four months of age. The caudoputamen is an exception, demonstrating a volume increase in Mbnl1+/−/Mbnl2−/− brains, we tested if Mbnl1 and Mbnl2 gene-specific effects are important variables in the maintenance of the structural integrity of the mouse brain. For this analysis, we studied female cohorts of 129sv Mbnl1−/−, Mbnl2−/−, Mbnl1−/−/Mbnl2+/−, and control Mbnl1+/+/Mbnl2+/+ mice at two months of age (n = 5/genotype). Two-month-old mice (2mo) were examined for this comparative study as 129sv Mbnl1−/−/Mbnl2+/− mice do not generally survive past the age of two months and because a significant attrition of Mbnl1−/− mouse cohorts occurs between the age of 2 and 4 months due to a sudden-death phenotype, presumably from cardiac arrhythmias32.As extensive changes in white- and gray matter volumes are observed in 4-month-old female Mbnl1+/−/Mbnl2−/− mouse cohorts. The Mbnl1−/−/Mbnl2+/−montages demonstrated the most obvious differences, containing in some cases 2–4 fewer slices, when compared with 2-month-old Mbnl1+/+/Mbnl2+/+ control brains   revealed a significant increase in apparent ventricle volume  and an enlarged apparent ventricle volume to whole-brain volume ratio  in Mbnl1−/−/Mbnl2+/− brains when compared with Mbnl1+/+/Mbnl2+/+ brains  Fig. ; Table 2ice Fig. ; Table 2ins Fig. ; Table 2p = 0.0008] and the corpus callosum/external capsule regions  was observed in the 2-month-old cohorts. For the corpus callosum/external capsule, post hoc Dunnett’s multiple-comparison test demonstrated comparable shrinkages in the Mbnl1−/−  and the Mbnl2−/−  mice, with the volume decrease being more prominent in the Mbnl1−/−/Mbnl2+/−  mice when compared with age-matched Mbnl1+/+/Mbnl2+/+ controls  and the Mbnl1−/−/Mbnl2+/−  mice, but not the Mbnl1−/− mice, demonstrated significantly reduced anterior commissure volumes when compared with 2-month-old Mbnl1+/+/Mbnl2+/+ control mice  = 9.370, ols Fig. ; Table 2ice Fig. ; Table 2p = 0.3004] , in the Mbnl2−/−  and in the Mbnl1−/−/Mbnl2+/− mice   = 1.327, 04] Fig. ; Table 272) Fig. ; Table 231] Fig. ; Table 2p = 0.3703]  and in Mbnl1−/−/Mbnl2+/−  brains when compared with age-matched Mbnl1+/+/Mbnl2+/+ brains  = 1.120, 03] Fig. ; Table 2ins Fig. ; Table 2t-test showed a significant volume reduction in the cerebellum of the Mbnl1−/−/Mbnl2+/− mice  but not in the Mbnl1−/− or the Mbnl2−/− mice, when compared with age-matched Mbnl1+/+/Mbnl2+/+ control mice  and increased apparent ventricle volume (~126% increase) and apparent ventricle volume to whole-brain volume ratio (~152% increase) when compared with controls. In the 2-month-old cohorts studied, Mbnl1−/−/Mbnl2+/− mice, but not the Mbnl1−/− and the Mbnl2−/− mice, show alterations in whole-brain (~6% decrease) and ventricle volumes (~53% increase)  and Mbnl2−/− mice (~31% decrease). Two-month-old Mbnl1−/−/Mbnl2+/− mice show a more prominent defect, with an ~43% decrease in the corpus callosum/external capsule volume  in the corpus callosum/external capsule volume when compared with control mice. These observations are consistent with a previous study demonstrating a role for cytoplasmic Mbnl1 in neurite outgrowth and development33. Four-month-old Mbnl1+/−/Mbnl2−/− mice show a less severe change (~21% decrease) in the corpus callosum/external capsule volume. It is unclear if this decrease in severity reflects possible compensation occurring in the corpus callosum/external capsule with age.A second prediction implicit in this hypothesis is that brain defects manifesting in DM1 patients encoding both small and large CTG tracts must be sensitive to smaller reductions in MBNL1 and MBNL2 levels. White matter defects are a widely reported pathology in both adult-onset DM1 patients, who have relatively small CTG tracts, and in congenital DM1 patients, who tend to have longer CTG tractsume Fig. ; Table 2Mbnl2−/− mice show a significant decrease in anterior commissure volumes (~31%), while 2-month-old Mbnl1−/− mice do not. Two-month-old Mbnl1−/−/Mbnl2+/− mice show a comparable phenotype to Mbnl2−/− mice, with (~29% decrease). Consistent with the stronger requirement for Mbnl2 in maintaining the structural integrity of the anterior commissure, 4-month-old Mbnl1+/−/Mbnl2−/− mice show a severe loss in anterior commissure volumes when compared with controls (~49%) , the hippocampus (~19%), the inter-/midbrain (~15%), and the hindbrain (~18%).Gray matter defects are observed frequently in the cortex, hippocampus, basal ganglia, striatum, thalamus, nucleus accumbens, cerebellum, and diencephalon in DM1 patientsMbnl1−/− (~12%) and not Mbnl2−/− mice show significant hindbrain-volume decreases. Strikingly, hippocampus volumes appear to be very susceptible to the loss of either Mbnl1 or Mbnl2 and all three 2-month-old cohorts, Mbnl1−/− (~13%), Mbnl2−/− (~14%), and Mbnl1−/−/Mbnl2+/− (~15%) show comparable hippocampal-volume losses. Thus, Mbnl1/2 depletion results in widespread gray matter volume reductions in the mouse brain in a manner reminiscent of DM1 patients  requiring both the loss of Mbnl1 and the partial depletion of Mbnl2. Hindbrain integrity is more sensitive to reductions in Mbnl1, as only Mbnl1+/−/Mbnl2−/− mice. This brain region is therefore more resistant to Mbnl1- and Mbnl2-dosage changes and appears to require a substantial decrease in total Mbnl1 and Mbnl2 levels to result in shrinkage. As cortex-volume decreases are observed in DM1 patients with small and large CTG tracts24, this observation suggests that alternate mechanisms may work with Mbnl1 and Mbnl2 loss to enhance cortex-volume reductions in DM1. Similarly, although gray matter atrophy is reported in the caudate nucleus and putamen in DM1 patients24, we observe an increase of ~7% in the caudoputamen in Mbnl1+/−/Mbnl2−/− mice. Other CUG-expansion RNA-mediated mechanisms must therefore be important in the development of pathology in this region.Isocortex-volume decreases (~8%) are observed only in 4. Impaired visuospatial learning and memory is pervasive in DM1 patients encoding a wide range of repeat sizes37. Consistent with this observation, we observe that the hippocampus, a region that contains a cognitive map of space, and which plays a role in learning and memory39, is particularly sensitive to alterations in Mbnl1 and Mbnl2 dosage. Thus, correlative changes in hippocampal-volume decreases and impaired visuospatial learning and memory are observed in the Mbnl1−/− and the Mbnl2−/− mice30. White matter volume decreases are observed in Mbnl1−/− brains and increase in prominence in Mbnl2−/−, Mbnl1−/−/Mbnl2+/−, and Mbnl1+/−/Mbnl2−/− brains  where the ATG encoding Mbnl1 exon 2 is deleted32. Development of 129sv Mbnl2ΔE2/ΔE2 mice (indicated as Mbnl2−/−) where the ATG encoding Mbnl2 exon 2 is deleted, is described in Mbnl1ΔE2/ΔE2/Mbnl2+/ΔE2 and 129sv Mbnl1+/ΔE2/Mbnl2ΔE2/ΔE2 mice denoted here as Mbnl1−/−/Mbnl2+/− and Mbnl1+/−/Mbnl2−/− mice, respectively. Age- and gender-matched wild-type 129sv mice (indicated as Mbnl1+/+/Mbnl2+/+) were used as controls. Mbnl1+/+/Mbnl2+/+, Mbnl1−/−, Mbnl2−/−, Mbnl1+/−/Mbnl2−/−, and Mbnl1−/−/Mbnl2+/− 129sv female mice at 2 and 4 months of age were used in this study. We have complied with the relevant ethical regulations for animal testing and research as specified by protocol number 11970, approved by the Institutional Animal Care and Use Committee of the University of Southern California.Mice were housed in standard cages on a 12 h light/dark cycle with access to water and a standard laboratory diet All MRI scans were performed using an MR Solutions  7T MRI-PET scanner using a 20 mm internal-diameter quadrature bird cage mouse head coil. The scanner is capable up to 7T and has ~24 cm bore size, with up to 600 mT/m maximum gradient. Mice were anesthetized with 2% isoflurane at flow rate 250 μL/min using . Animals were transferred onto a temperature controlled heated scanner bed  with anesthesia levels maintained between 1.5–2%. Temperature was monitored using a fiber-optic thermocouple and maintained at 37 °C. A pneumatic pillow was placed underneath the animal for respiration monitoring. Mice were positioned at the magnet-bore isocenter using a motorized system. Temperature and respiration were monitored by the accompanying PC-SAM software . Animals were placed in a heated recovery chamber after the scan, until the animals were ambulatory. A gradient echo scan was used to obtain three orthogonal slices for positioning followed by 2-dimensional (2D) fast spin-echo (FSE) T2-weighted (T2w) sequences to define neuroanatomy in the transverse axial orientation. FSE T2w scan parameters were as follows: TE = 45 ms, TR = 4000 ms, number of averages = 4, echo-train length = 7, field of view = 14 mm × 14 mm, slice thickness = 0.5 mm, number of slices = 28, and matrix size = 256 × 256.40 and using Hof et al.41 and the Allen mouse brain atlas as visual guides (http://mouse.brain-map.org/). ROI area measurements for each slice were multiplied by the image-slice thickness (0.5 mm) and summed to generate volumes. Three blinded experimenters  independently drew all ROIs and calculated volumes. Statistical analysis of volume measurements demonstrated no significant differences between the experimenters. Discussions were held by the experimenters to reach an unequivocal consensus on disparate measurements. The following white and gray matter ROIs were measured: defined white matter regions included the anterior commissure and the corpus callosum/external capsule. Gray matter regions included the cerebrum , brainstem (inter-/midbrain and hindbrain), and the cerebellum. Whole-brain and apparent ventricle volumes were measured. Apparent ventricle volumes were delineated as visibly identifiable ventricle regions with hyperintense signals in the FSE T2w brain slices. The olfactory bulb was excluded in volume measurements.Regions of interest (ROI) were manually defined on the 2D T2w FSE MRI scans using the polygon tool in ImageJ softwareTM ECL plus western blotting substrate (Thermo Scientific) according to the manufacturer’s protocol and the signal was detected on a ChemiTM Doc MP imaging system (Bio-Rad). Anti-Gapdh and anti-Mbnl2 antibodies were purchased from Santa Cruz Biotechnology ), anti-Mbnl1 antibodies are a gift from Ian Holt, dilution 1:10042. Uncropped Western blot images are shown in Supplementary Fig. Cortex, hippocampus, cerebellum, mid-/hindbrain regions, and all other brain regions, including interbrain and caudoputamen, were dissected and flash-frozen in liquid nitrogen. Frozen brain samples were lysed with modified RIPA buffer  and equal amounts of protein (15 ug) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk in TBST , for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight with rocking. The membranes were subsequently incubated for 2 h at room temperature with secondary antibodies conjugated with horseradish peroxidase. After washing with TBST, the membranes were incubated with PierceMbnl1+/+/Mbnl2+/+ and Mbnl1+/−/Mbnl2−/− cohorts. One-way ANOVA analysis was performed when comparing the ROI volumes (mean ± SD) of 2-month-old Mbnl1+/+/Mbnl2+/+, Mbnl1−/−, Mbnl2−/−, and Mbnl1−/−/Mbnl2+/− mice, followed by the Dunnett’s multiple-comparison test. In some cases, the two-tailed t-test was performed when comparing genotypes in the 2-month-old cohorts. GraphPad Prism 9  statistical software was used and a value of p < 0.05 was defined as statistically significant. For all 4- and 2-month cohorts, n = 5 mice/genotype.Two tailed t-test was performed when comparing the ROI volumes (mean ± SD) of 4-month-old Further information on research design is available in the Transparent Peer Review FileSupplementary MaterialDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Cannabis genus. Its non-psychoactiveness and therapeutic potential, partly along with some anecdotal—if not scientific or clinical—evidence on the prevention and treatment of neurological diseases, have led researchers to investigate the biochemical actions of CBD on neural cells. This review summarizes the previously reported mechanistic studies of the CBD actions on primary neural cells at the in vitro cell-culture level. The neural cells are classified into neurons, microglia, astrocytes, oligodendrocytes, and neural stem cells, and the CBD effects on each cell type are described. After brief introduction on CBD and in vitro studies of CBD actions on neural cells, the neuroprotective capability of CBD on primary neurons with the suggested operating actions is discussed, followed by the reported CBD actions on glia and the CBD-induced regeneration from neural stem cells. A summary section gives a general overview of the biochemical actions of CBD on neural cells, with a future perspective. This review will provide a basic and fundamental, but crucial, insight on the mechanistic understanding of CBD actions on neural cells in the brain, at the molecular level, and the therapeutic potential of CBD in the prevention and treatment of neurological diseases, although to date, there seem to have been relatively limited research activities and reports on the cell culture-level, in vitro studies of CBD effects on primary neural cells.(‒)-Cannabidiol (CBD) is one of the major phytocannabinoids extracted from the  R,6R)-3-methly-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol, CBD) is a major non-psychoactive phytocannabinoid derived from the Cannabis genus. CBD has emerged as a ray of hope for neurological disorders in pharmacological and medicinal fields, because of its deep and wide potential of therapeutic effects including neuroprotective, anti-inflammatory, anti-epileptic, anxiolytic, anti-depressant, and even anti-addictive effects ..23]. Thewith CBD . The rep2O2), a pivotal mediator of cellular oxidative stress, for primary cerebellar granule neurons (CGNs) i) has been reported to be controlled mainly by the ion flux, via plasma membrane receptors, voltage-gated ion channels, and the intracellular storage in the organelles, such as mitochondria and ER i changes upon the CBD treatment of mature hippocampal neurons under various conditions [2+]i of the neurons in the physiological buffer solution  by about 45% compared with the basal [Ca2+]i, and any [Ca2+]i increase-induced neurotoxicity was not observed for 48 h [2+]i tended to persist even after cell washing, and only 19% of the neurons fully recovered [Ca2+]i after two consecutive CBD treatments with an interval of 15 min. In addition, the [Ca2+]i increase in the CBD-treated neurons was not blocked completely by either cadmium, a non-specific VGCC blocker, or nifedipine, a selective blocker of L-type VGCC; in both cases, the [Ca2+]i increase was measured to be 18%. These results suggested that the CBD-induced [Ca2+]i increase in hippocampal neurons might involve the Ca2+ release from the intracellular stores  as well as the calcium gradient and L-type VGCC. Another report showed that CBD (10 µM) led to the immediate [Ca2+]i increase even in the Ca2+-free saline, supporting the potential participation of the intracellular stores in the [Ca2+]i regulation of CBD [2+]i increase was reduced in the highly excitatory buffer that contained Ca2+ and potassium (K+) ions 3–3.5 times more than the standard buffer, implying that the extracellular space might not be the major source in [Ca2+]i regulation by CBD.Platt et al. studied the regulatory roles of CBD in the intracellular Canditions ,28,30,312+]i with the transient or delayed downregulation of mitochondrial Ca2+ concentrations, based on the co-imaging data made with a cytosolic Ca2+ indicator, fura-2 AM, and a mitochondrion-specific Ca2+ sensor, Rhod-FF, AM  was blocked fully on average, when hippocampal neurons were pre-treated with thapsigargin (Tg) (2 µM), an irreversible blocker of ER Ca2+-ATPase, which causes the depletion of Ca2+ in ER and prevents any subsequent occurrence of ER-dependent responses [2+]i increase that was induced by Tg (200 nM) i might be associated with the actions of ER as a source of Ca2+. Relatedly, NAGly, an endogenous cannabinoid, which also depressed SOCE, induced the increase in cytosolic zinc (Zn2+) level, implying the potential regulation of CBD on the Zn2+ signaling, which is associated with learning and memory defects [In addition to the reports on the mitochondrion-mediated [Cae action . For exaesponses . Another(200 nM) . The CBD defects .2+]i-regulatory action of CBD was also involved in the endogenous cannabinoid system. CBD was known to upregulate the endogenous N-arachidonoylethanolamine (AEA) level by preventing its re-uptake and hydrolysis [2+]i in several cellular models [2+]i by 45%, but the increase dropped to 22% or 12%, respectively, upon the co-addition of AEA or 2-AG with CBD. In addition, the pre-treatment of the neurons with glutamate decreased the CBD-induced [Ca2+]i elevation to 26% (from 45%). The results suggested that the endocannabinoid-dependent effect of CBD on [Ca2+]i might involve the direct, inhibitory action of the endocannabinoids and/or the activation of specific receptors, but the experiment showed that the increased level of the endocannabinoids, which was made by the addition of enzyme inhibitors of AEA-hydrolyzing FAAH and 2-AG-hydrolyzing MAGL, did not alter the [Ca2+]i increase induced by CBD. It was reported that the i increase in hippocampal neurons, which implied that the CBD action on [Ca2+]i was regulated negatively by CB1 and TRPV1 i increase, while the CBD-induced [Ca2+]i response was greatly increased to 312% from 45% by a PLC inhibitor (U73122) [q/11/PLC signaling might be related negatively with the [Ca2+]i-modulatory action of CBD. Drysdale et al. reported that the antagonists of CB1 i.e., AM281) and TRPV1  enhanced the CBD-induced [Ca1 and TRP2+]i increase in hippocampal neurons [2+]i enhancement was 45% or 58% by CBD or THC only, respectively, the 1:1 mixture of CBD and THC increased [Ca2+]i by 189% (for the mixture of purified compounds) and 314% (for the mixture of extracted compounds) with highly increased proportion of the responsive cells. CBD and THC seemed to work on the [Ca2+]i regulation via different mechanisms, but the synergistic action could support the reports that Cannabis extracts had additional benefits in several clinical studies i homeostasis. Correspondingly, mitochondrial dysfunction has been known to be related with various aging-induced neurodegenerative diseases, such as AD, PD, and ALS i homeostasis. However, cannabidiolic acid (CBDA)  , an acidAnother report showed that the neurotoxicity of rotenone on TH-specific, dopaminergic neurons in the mesencephalic culture was ameliorated by CBD (10 µM), with the viability increase to 42% from 29% . The aut®, a CBD extract, for epileptic seizures i homeostasis, migration, and apoptotic process—and the therapeutic potential of CBD for neurodegenerative diseases by blocking the detrimental, self-accelerating glia-inflammatory cycles  to 140% in the primary microglial culture, which was more efficient than other cannabinoids, WIN 55212-2 and JWH-133  75]. It. It75]. In addition to the inflammatory modulation of CBD under neuropathological conditions, CBD was suggested to directly attenuate microglial activation ,76. For i/o-linked GPCRs, including CB1 and CB2, were suggested not to be involved in the CBD-based regulation of microglial phagocytosis, based on the following observations: (1) PTX did not affect the CBD-induced phagocytic activity; (2) CB1/CB2-agonistic phytocannabinoids did not increase the phagocytic activity; (3) CBD induced the increase (161%) of phagocytosis for CB1/CB2-deficient BV-2 cells. On the other hand, CBD-induced phagocytic increase was silenced in the extracellular Ca2+-absent environment made by EGTA, but not in the intracellular Ca2+-deficient condition by BAPTA-AM, implying the potential role of Ca2+ signaling in the CBD-based regulation of microglial phagocytosis. In addition, the phagocytic activity, enhanced by CBD, was reduced with various transient receptor potential (TRP) channel blockers—ruthenium red (a non-selective TRP channel blocker), SKF96365 (a TRPC and TRPV blocker), and CPZ or AMG9810 (both TRPV1 antagonists). The phagocytic modulation of CBD was identified to be mediated by the activation of TRPV2 through the quickly stimulated expression of TRPV2 and its promoted translocation to the plasma membrane by CBD. However, the observed different [Ca2+]i responses of primary microglia and BV-2 cells, upon CBD treatment, would necessitate further mechanistic studies with a focus on primary microglia: while CBD (10–1000 nM) could not change [Ca2+]i in primary microglia [2+]i by 2.43-fold, and the increased [Ca2+]i was sustained even after washing in the case of BV-2 cells i homeostasis might proceed through both cannabinoid-dependent and -independent pathways  promoted the LPS-induced migration of primary microglia with a 2.2-times increase under physiological conditions in the CB1 and CB2-dependent manners . Anotherof 25 nM . HoweverThe induction of apoptosis of over-activated immune cells is one direction of immunomodulation or immunosuppression. CBD has been suggested to stimulate the apoptosis of immune cells, such as lymphocytes and monocytes ,91, as wAn astrocyte is the largest proportional glial type in CNS. They widely provide physiological supportive functions for neurons, such as the provision of nutritional, metabolic, and structural supports, regulation of extracellular ion homeostasis, and assistance to synaptic function with the control of neurotransmitters ,96. In nCBD has been shown to play a therapeutic role in inhibiting reactive astrogliosis in various in vivo models of brain disorders. For instance, CBD reduced the GFAP-overexpressed reactive astrocytes in the pilocarpine-induced epileptic model through the activation of the PI3K signaling pathway . CBD als®, a 1:1 combination of CBD and THC, inhibited the over-production of CSPGs  from 236% to 95% and normalized the upregulated mRNA expression of core components in the CSPG synthesis  in the TGF-β1/bFGF-stimulated astroglial culture. These results indicated that CBD provided an environment to permit axonal regeneration and myelin repair by interfering with the accumulation and production of CSPGs. In addition to the regulation on inflammable gliosis, CBD was reported to universally modulate the entire immune system, including leukocytes, endothelial cells, microglia, and astrocytes. It suppressed the transmigration of leukocytes into the brain by reducing the production of VCAM-1 in endothelial cells and chemokines  in lymphocytes and astrocytes, as well as preventing the microglia activation in the TMEV-induced, demyelinating disease model . In the The immunomodulatory actions of CBD were investigated in the co-culture systems of several cellular members in CNS, including neurons, microglia, astrocytes, and endothelial cells. CBD (5 μM) attenuated the PrPres toxicity, with viability increase from 37% (without CBD) to 69% (with CBD), in the PrPres-infected neurons co-cultured with N11 cells—non-primary microglial cells, implying that CBD might control the inflammatory responses of reactive microglia triggered by the PrPres deposit, such as the secretion of neurotoxic factors . The antThe exact operational mechanisms of CBD  remain unresolved. In this regard, it would be necessary to investigate the complex actions of CBD in the co-culture system of primary cells from the nervous system and get an insight into how CBD regulates the collaborating and stimulating actions in the nervous system. Auzmendi et al. investigated the effects of CBD on the neurovascular unit, which was composed of astrocytes and vascular endothelial cells and regulates the homeostasis of hemodynamics and controls the access of drugs into the brain . They obThe oligodendrocyte is another neuron-supporting glial cell in the CNS. Oligodendrocytes not only produce the axon-wrapping myelin sheath, enabling the rapid and long-distant transfer of action potentials, but also maintain ion homeostasis and supply trophic factors. During development, oligodendrocytes are generated from oligodendrocyte progenitor cells (OPCs), but a large portion of OPCs remains undifferentiated and immature as a potential repository for remyelination . Oligode2+]i homeostasis by CBD but not to receptor-mediated pathways , based on the observation that the cytotoxicity of CBD was reduced only in the depletion of extracellular Ca2+. In a similar trend to the aforementioned induction of neuronal [Ca2+]i increase by CBD [2+]i by 43% in the non-specific cortical glial cells. The [Ca2+]i increase in the glia was observed to be made through L-type VGCC and intracellular Ca2+ stores, but it was negatively controlled by CB1 and TRPV. In addition, the proportion of CBD-reacting cells was higher for glia than for neurons [2+]i increase in primary oligodendrocytes in a concentration-dependent manner, and the increase was blocked partially by the elimination of extracellular Ca2+ or mitochondrial impairment, not by the blockades of CB1/2, TRPV1, L-type VGCC, ryanodine, and IP3 receptors [2+]i increase in oligodendrocytes.CBD (>0.1 µM) increased the death rate of primary oilgodendrocytes, while primary cortical neurons did not show any cytotoxic results with 10 µM of CBD ,78. In ae by CBD ,28,29,30eceptors . The CBDeceptors . In addi2+]i increase, nor alterations of the cell cycle and proliferation rate for primary OPCs 113]. The2+ signaling activity. For example, all the CBD-treated cells increased [Ca2+]i immediately as a response to the KCl stimulation, which induces the Ca2+ influx into the cytosol via VGCCs [2+]i increase slightly less than the vehicle control. Furthermore, only 8.7% of the CBD-treated cells reacted to the histamine stimulation, which induces the [Ca2+]i increase through the histamine receptors that are known to be expressed mostly in immature neurons. The 37-day CBD treatment increased the expression levels of CNR2, encoding the CB2, and GAD67 (a marker of GABAergic neuron), and it decreased that of VGLUT1 (a marker of glutamatergic neurons).On the other hand, Bekman et al. reported that CBD neither promoted nor inhibited the neuronal differentiation and maturation of the NPCs that were differentiated from ihPSCs . The CBDia VGCCs , with thCannabis genus and identified, but one of the two main phytocannabinoids, CBD and THC, which have unceasingly attracted profound attention and interest from researchers as well as the general public from the aspect of their potential health benefits. Compared with THC that is psychoactive, non-psychoactive CBD has been on the market in various forms  for cosmetic and “allegedly nutraceutical” products in some countries. CBD has been claimed to have many health benefits, such as pain and anxiety relief, symptom alleviation for epilepsy and cancer, management of neurodegenerative diseases, and acne treatment, but many of them have not so far had solid scientific data for supporting the suppositional benefits [®), as a prescription medicine for the treatment of seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, or tuberous sclerosis complex in patients 1 year of age and older, has boosted the interest in CBD as a potential therapeutic candidate for other diseases, including neurodegenerative diseases. Moreover, diverse drugs containing CBD in various formulations have been proposed or are actively under development, such as Sativex®, an oromucosal spray composed of CBD and THC, for the treatment of spasticity and pain related to MS [TM (ZYN002), a CBD-formulated transdermal gel for the treatment of Fragile X syndrome [CBD is not just one of the 100+ compounds extracted from the ed to MS ,124, PTLed to MS ,126, andsyndrome ,128.This review has focused on the biochemical actions of CBD in neural cells at the cell-culture level in order to provide an overview of the research efforts for a molecular level, mechanistic understanding of how CBD affects the neural-cell behaviors under normal and pathological conditions . DiscussCannabis and the low amount of pure CBD extracted from Cannabis. The interdisciplinary nature of CBD studies also might deter the in-depth fundamental research on CBD effects on neural cells in the brain, because it requires the synergistic contributions from neuroscience, medicinal chemistry, biochemistry, biology, and bioinformatics to name a few.However, the limited number of research articles has so far been published on in-vitro culture-based studies of CBD actions on neurological diseases and others, presumably because of the legal scheduling of Cannabis plant, which would defer the mass production of CBD at its highly pure form. Mass production will be beneficial to the fundamental study of the CBD action not to mention for its medical use at the market. Therefore, it is recommended to develop alternative methods for the mass production of pure CBD. Total synthesis would be one of the strategic options [CBD production has relied mainly, if not solely, on the natural source,  options . Not onl options , which cIt is anticipated that detailed understanding, at the fundamental level, with solid experimental data, in the combination with the results from (pre)clinical studies, would solidify the path to the medical use of CBD. For example, the biological actions are complex and intertwined, and its network is highly interconnected in the many-to-many mode, although this review has primarily dealt with the CBD actions on each cell type in the neural cells separately. Further studies on cell-to-cell interactions induced by CBD would give more mechanistic information at the molecular level. Contributions from other domains also could be made, such as the fabrication of artificial tissues or organoids in biological and biomedical engineering. The cellular construct is innately three-dimensional (3D), but most of the previous studies discussed in this review have heavily relied on the 2D culture, which might not faithfully recapitulate the cellular states in the brain in vivo from the chemical, biological, and mechanical viewpoints ,132,133.


The importance of embodiment for effective robot performance has been postulated for a long time. Despite this, only relatively recently concrete quantitative models were put forward to characterize the advantages provided by a well-chosen embodiment. We here use one of these models, based on the concept of relevant information, to identify in a minimalistic scenario how and when embodiment affects the decision density. Concretely, we study how embodiment affects information costs when, instead of atomic actions, scripts are introduced, that is, predefined action sequences. Their inclusion can be treated as a straightforward extension of the basic action space. We will demonstrate the effect on informational decision cost of utilizing scripts vs. basic actions using a simple navigation task. Importantly, we will also employ a world with “mislabeled” actions, which we will call a “twisted” world. This is a model which had been used in an earlier study of the influence of embodiment on decision costs. It will turn out that twisted scenarios, as opposed to well-labeled (“embodied”) ones, are significantly more costly in terms of relevant information. This cost is further worsened when the agent is forced to lower the decision density by employing scripts . This adds to our understanding why well-embodied (interpreted in our model as well-labeled) agents should be preferable, in a quantifiable, objective sense. Research in robotics, while dominated by kinematic and dynamic control laws, has increasingly identified the importance of embodiment for effective operation . DespiteMuch of the historical discussion and motivation for studying good embodiment comes from studying biological examples . Living In recent years, several attempts have been made to formally incorporate and measure the impact of embodiment on information processing in artificial agents. In , the autreflexes and fixed action patterns  neural control without the involvement of higher brain areas . Many reSimilarly, fixed action patterns require little or no sensory input and are found often in biological organisms. Such behaviors comprise complex movements across a whole species that vary only in intensity (decided at the start of the movement) but not in form . One exaUnder the assumption that organisms attempt to minimize information processing , fixed aConcretely, we approach action sequences by combining already existing (basic) actions into larger “super”-actions that do not require sensor input while running and are thus similar to the behaviors of fixed action patterns. These are what we call “scripts”. We then investigate the efficacy of these action scripts using the information-theoretic framework from  and inveThis work is organized as follows. A, S, lower case letters to define a concrete value a, s and S to assume a value S and t (and s and t is not of importance.We use uppercase letters for random variables We use Markov Decision Processes (MDPs) to model the perception-action cycle assuming that the world state can be—in principle—fully known.a in state s and ending in state s and action a chosen.For the definition of an MDP we follow . Given aa to choose in the state s. Following the behavior specified by the policy, the agent will accumulate a reward depending on the state A policy is denoted as follows We will assume episodic tasks throughout the paper. Therefore, we do not need to include a discount factor in the cumulated reward . We see the policy that maximizes the future expected return  distance from s to g  defined as:X and Y. This quantity is non-negative and vanishes if and only if X and Y are independent, that is when X does not provide any information about Y and vice versa. For further details see  actions. These actions vary depending on the task and the granularity of the model . Here weWe define scripts as a sequence of This defines a single script as an ordered tuple, or better, a sequence of all its components, for example a falcon .The new actions space in our model contains all basic actions and all scripts up to a certain length: The scripts now modify the Bayes Network of the perception-action loop. As in a perception-action loop with no scripts available, the state of the world changes every time step, depending on its previous state and the basic action performed by the agent see , top. ThWith this, the intermediate layer in the form of scripts, this layer now takes care of the sequence of actions and the timing of when to perform these. We emphatically shall not consider the processing and management cost required for running the script itself once it started and assume the necessary hardware (such as in the form of a suitable control circuit) is part of the agent’s given design.With the basic actions unchanged, scripts containing more basic actions take more time steps to complete than shorter scripts or basic actions. Thus, scripts provide a form of temporal abstraction.As a consequence, this forms a semi-MDP as the time steps of the world no longer match the time steps  of the agent. For simplicity reasons, we will not model the length of the scripts in time. Instead, we will only model the time steps when the decision of the agent occurs and hide the difference in “world-time-steps” in the reward  function: A script simply gives as much reward as if all basic actions it contains were executed in sequence, implying it took longer. From the agent’s perspective, this is just a different cost but with one time step. Effectively, the agent is only aware of those time steps in which it has to decide, even though the world itself has finer time steps. Hence, by only modeling the time steps of the agent, we can treat it as an MDP instead of a semi-MDP.Considering the scripts plainly as extensions of the action space, they all follow the definition of an action in the traditional MDP-framework with all state transitions being defined as With scripts available, every path can either be achieved by a script moving along the full path or shorter scripts and basic actions each only moving along part of the path. This is also true for every optimal path. Hence, the resulting value-optimal policies exhibit multiple optimal actions (basic action or scripts) in every state and could be probabilistically selected even though they might use the same basic action, whether on its own or inside a script.The new action space is also significantly bigger. In fact, the potential action space can grow exponentially with the maximum script length permitted. Hence, the computation time will thus grow as well. This renders the optimization over the whole action space that would contain every possible script infeasible for longer script lengths. This also makes identifying ‘good’ and ‘bad’ scripts and preselecting the relevant actions and scripts in the action space accordingly an important topic for the future.The state space of an MDP does not have an initial interpretation as anything else than a set of discrete, a priori unrelated states. Linking these states using labeled actions often introduces additional semantics in the labels of the states and actions. In typical scenarios, e.g., in navigation, the same action label always leads to the “same”  change of the state. This injects a semantics into the labeling which is not part of the core MDP formalism. The goal of the twisted world, defined by , and subembodied agent, though , the agent “takes its actions with it” . To reach the goal, this identifier is what the agent has to look for, and thus constitutes the information it needs to process.Different agents can have different action labels while existing in the same world and having the same goal, making them look for different clues in the world. Two hikers could follow the exact same path, one using a globally oriented map and a compass to follow the global properties while the other uses the local signposts. The decision of whether to follow choose the next path based on the global direction or follow the signposts critically depends on the particular choice of embodiment. In a way, the coherent label arrangement in global navigation can be considered a well-adapted choice of labeling or an embodiment. It corresponds to a well-chosen link between action labels and the effect this has on the world dynamics or the task.additional assumption which we—in our model—aim to study separately. We will now construct a formalism that enables us to separate out the implicit assumptions made in typical MDPs, especially those embedded into a geometrical context, which is called “Twisted World”.The agent’s choice of its internal labeling of its actions can thus contain information about the world or its task. As an external observer, we cannot make a definite assumption about the internal labeling. As an example, the action “going along a certain street” leading to the factory and the train station might have a different label for the factory worker (way to factory) and the commuter (way to station). In other words, a “natural” link between action label and the expected effect on the agent in its world may be present, but is always an Twisted World is a modification of a given MDP that aims to locally remap the action labels onto the transitions. During this process, we ensure that all actions lead to different transitions and all transitions remain feasible. Thus, in terms of action possibilities and under a traditional MDP perspective where each state s has its own—possibly singular—action set, the problem to solve the MDP remains precisely equivalent. However, it will turn out that this change has significant when we consider the informational cost of the resulting policies, as these look substantially different after the remapping  of a twisted world to be the ratio of the number of twisted states We now define the control + z might mean undo in both programs, another shortcut might mean to different things, such as control + d meaning delete in program one while meaning duplicate in program two.The twistedness of the world shows how much of one state’s perception-action loop is transferable to the other states. An intuitive example for a partly twisted embodiment is how keyboard shortcuts change their meaning when a human switches from one software to another one. While Twisting the world allows for an interesting anomaly: perfect twists. A perfectly twisted world is a world in which the agent can follow just one action label to reach the desired outcome we assume to be fixed. In a world in which all actions are linked to the global directions, the agent cannot move into different directions following just one action label. In a twisted world, this is possible. Carefully selected twists can create direct paths leading the agent to the desired outcome from any state using just one action label.In randomly selected twists, however, it is, of course, highly unlikely that such a perfectly goal-adapted twist occurs. On the other hand, in nature, we observe many behaviors that can be derived by following a simple gradient or feature of a sensor stimulus such as optical flow, angle to a light source for flying insects or similar . In thisIn our examples, we limit ourselves to—in principle—fully observable worlds, so the sensor does not contribute to the simplicity of the action selection. In this sense, the perfect twists are the perfectly adapted embodiment, purely in terms of action labeling, to the world and the task.Introducing scripts allows an agent to make a single decision and multiple actions. Hence, the agent can execute actions without deciding on it in every time step of the world. This allows us to pinpoint at which states in the world the agent takes a decision and where it does not. This further, allows us to model the cost of a decision only when one occurs. We consider two aspects when putting a “cost tag” on decisions, one is our informational costing of decision-making, and secondly, achieving a more refined analysis also permits the inclusion of an explicit cost for taking a decision at all. This will be a cost, expressed in value units which encompasses the sensory cost, information processing, and the process of initiating the decision itself. Note, this cost is separate from the informational treatment and we will model it as an abstract cost in the reward function. As in other mentioned work, the informational cost of the decision will, in contrast, be quantified in terms of Shannon information using the relevant information formalism .To define the cost of a decision we first define “decision” in our context. Usually, people using the term “decision” mean the trigger point of a plan or an action after considering multiple possibilities . The infAn agent in the MDP framework needs to decide on an action based on the state it is in and thus needs to know in which state it is. Thus, the agent needs a certain amount of information about which state the current state is. Essentially, the cost is expressed here as the “per-step” relevant information that the agent needs to process about the current state to select the appropriate action which can be seen as the actual coding cost for the message sent from the sensor to the actuator . When anrelevant information of an MDP has been defined as the minimal amount of Shannon information required about the current state to select an action and achieve a given average utility s, taking action a. Therefore, relevant information finds the policy with the smallest mutual information among all policies achieving the optimal value.Formally, the [Q] . As the This is a constrained optimization, which we solve by introducing Lagrangian factor β:E is taken over the joint distribution of all states S and all actions A given by β needs to tend to the infinite limit to achieve optimality of the value. The expectation  problem  with a d problem  to solveecessary . If the Throughout this paper, we will assume a uniform distribution Knowing when decisions occur allows us to analyze them further. We do this by introducing a new component to the reward-function, which depends on the occurring decisions. Combining both rewards, we get a new reward function of:This function allows to create a cost for a decision in addition to the cost of performing an action. We will use this reward function throughout the paper where, depending on the study, we will set After introducing scripts as an extension to the action space for RL we will investigate how this changes the agents’ behavior in a simple grid-world navigation task how the cost of decision factored into the reward function will affect this.During the following experiments, we will consider a small grid world of We will sometimes model an explicit decision cost. In this case, we define the cost of every single decision to be −1 see . With thNorthern Border: This goal is composed of all northernmost states of the world. With only basic actions available, the relevant information is zero as the optimal action in all states is the same, going north.Central State: Only the central state, the state furthest from any border of the grid, is a goal state. Reaching the goal requires different actions from different sectors on different quadrants of the goal. Thus the relevant information is comparably high with 1.28 bits. Center Line: Here a whole line running through the center is set as goals. With only basic actions, the policy contains only two of the four different basic actions driving the agent toward the goal with a relevant information of 0.80 bits. Corner State: Here, the goal is one state in one of the corners of the world. The policy for this task contains two actions and a relevant information of 0.25. As with the “Center Line”-setup, we only require two actions to reach the goal, but one of the actions is far more likely than the other, to achieve a lower mutual information.We will look at four different goal setup, either single states or a set of states of varying informational hardness (for basic actions), which will permit exploring how different aspects of our  problem “morphology” changes affect the information balance of the problem. The individual goal state sets are shown in red in For the next experiments, we will remove the well-structured internal labeling of the actions by employing “twisted worlds” with varying degrees of twistedness  lead under a Zero-Information policy. More precisely, instead of specifying a goal, and computing the cost of an optimal goal-directed policy at minimum information, we take the converse approach: we select a Zero-Information deterministic policy and establish how close to potential target destinations the agent can get following these in a twisted world. The idea of this experiment is to measure how effectively an agent can traverse a world using no information, i.e. sensory input, that is, in open-loop mode.Formally, a Zero-Information policy is a policy for which the mutual information is zero While traversing the world in an open-loop manner given a fixed action, the agent follows a certain trajectory. We will characterize the coverage of the state space for these “skeleton” trajectories through the world using two different metrics for each potential goal state.s and possible goal g, we consider the trajectory generated by a deterministic Zero-Information policy and extract for each state on the trajectory how much the optimal cost  for each of these states is and also how far  one has to move to minimize this cost. In other words, if the generated trajectory (assuming the movement is deterministic) consists of the states The coverage of the Zero-Information trajectories is measured in the following way: For each state For this experiment we will use a grid world as before but this time of the size As general observation, the results show that, while scripts reduce the decision density, this only rarely does not incur additional informational costs while employing scripts usually either leads to a higher information cost in goal-directed tasks or leads to a loss in value when following open-loop trajectories. In twisted worlds, scripts are typically informationally more costly, but in rarer cases counteract the effect of the twists. We will start with the results comparing basic actions and scripts for goal-directed behaviors in well-structured worlds. Then, we proceed with the results in twisted world including a newly found phenomenon similar to the perfect twist which we will discuss below as “special twists”. Subsequently, we will present the coverage of worlds when using Zero-Information policies.For all experiments we only considered policies achieving the optimal value as defined in Adding scripts to the action space without adding a separate decision cost does not change the relevant information for any of the goal sets as compared to only having basic actions available. The optimized policies for the “Northern Border” setup and the “Center Line” setup both contain scripts. The policies for the “Central State” setup and “Corner State” setup which constitute more localized goals only use basic actions see . Adding For the experiments in twisted worlds, we considered the same world size and goal sets but every world contained a certain percentage of twisted states. We are interested in the relevant information and the occurrences of scripts in resulting policies, this time depending on the twistedness. Overall, the relevant information and the use of scripts becomes more similar across the goals with increasing twistedness of the world. The exact results are shown in The relevant information rises for three  of the four goals and only goes down for the last one  which is not affected by adding a decision cost. This means that the untwisted world is the “easiest” world for three of our setups and the hardest for the goal in the center. In particular, the untwisted world proves to be unsuitably twisted for this particular goal.Without a decision cost, scripts appear less often in the policies navigating the twisted world. Furthermore, the development of the relevant information for the “Central Line”-setup and the “Corner State”-setup is surprisingly similar. However the use of scripts is very different: The “Corner State”-setup uses scripts rarely, independently of the twists, while the “Central Line”-setup still uses scripts in the fully twisted world.Interestingly, we see that the goals containing five states are equally difficult in the fully twisted world which is also the case for the two goal setups containing only one state. Thus, showing goals consisting of an equal number of states seem equally difficult to reach in a fully twisted world.In the plot with decision costs, we see that all goals have a higher relevant information in a fully twisted world compared to those without it. Note, the scales of both plots are different. Since the agent is incentivized to minimize the number of decisions, every policy contains scripts. An interesting observation is that the “Northern Border”-setup (blue) which is not harmed by the decision cost in the untwisted world becomes the most difficult one in a fully twisted world. The “Center Line”-setup (cyan), initially not affected by the decision cost as well, shows a different behavior: It becomes the easiest setup in terms of relevant information on average at Generally, the development of the setups with and without decision cost appears similar, but, on closer analysis, one finds that the decision cost has a big impact on the behavior in twisted worlds.Interestingly, there are “special” twists allowing the agent to use scripts without affecting the relevant information compared to the relevant information when only basic actions are available. These particular scripts fully negate the effect of the north , concretely: green: An example of such a special twist for the “Northern Border” setup is shown in However, when those exact same twists do not occur all in the same row but are scattered in the world, the information-optimal policy only contains basic actions. It is important to note that this policy still has the same relevant information as with the ordered twists. Thus, we observe that scripts require more regularity to be used in an information-optimal fashion.The main objective of these experiments is to investigate the effect of twists on the coverage of the environment if a deterministic open-loop policy is run (either a single fixed basic action or a repeated application of a fixed script).The effect of this depends on both the twist and the action (basic action or script) selected. In general, we find that the scripts get to the other states in fewer decisions and that the distances in twisted worlds tend to be higher. The detailed results are shown in the following subsections.west), shown in blue (108), the script  shown in green (115), or the script  shown in magenta (116). Using any of these actions, the average distance to the possible goal states,  decreases when moving until the wall around the world is reached which stops further coverage. The basic action and the script  both move along the same trajectory. Both actions visit the ten states directly shown by distance zero as shown in the second and third plots of First, we compare a basic action to two different scripts shown in west, north). In the last plot of Now, we look at a script that actually includes different basic actions, a capability only possible after the introduction of scripts, here , shown in magenta (▼), and , shown in green (▲). Again the agent started in the southeast corner. Further, the basic action (in blue (●)) from five is added as a baseline. The first graph shows that the magenta (▼) action does not get any closer to the other states after the first executed step while the green (▲) one does. In west, east, west, east), in fact, this scripts only ever visited two states. We will refer to scripts with this behavior as “non-traversing” in the future. The script  on the other hand achieves smaller distances to many states than the basic action. In fact, from the third plot, we see that 20 states are directly visited by this script.Next, we compare two scripts we found to achieve a dissimilar coverage of the environment: In Both of these scripts are extreme cases found during the experiments with all scripts. Thus, the other scripts fall somewhere in between those extremes. This shows, however, that the different scripts are of highly different usefulness for traversing the world.Lastly, we compared scripts of different lengths using the average distances per step averaged over all scripts of a certain length and all states of the world as starting states. The blue (●) line in west, west, west) but also those which only effectively move one step such as . The second type of action traverses the world more slowly but actually improves the distances to some possible goals. Furthermore, there are no scripts of length three visiting only one or two states in all start states. Thus, we cannot prune low-performing scripts of length three as simply as with scripts of length two. All longer scripts behave similarly with the evenly long scripts including some non-traversing ones and the unevenly long scripts not.The magenta (▼) points refer to the average distances achieved with scripts of length three. With these the distances not only decrease in fewer steps but also decrease further compared to the scripts of length two. This is due to the fact that this action space includes very far moving actions such as ) is evaluated and actually becomes more viable with more states being twisted. It did not reach more coverage than the previously good script ) in The reason for this is shown in The achieved distances of the bad script show it is getting better the more the world is twisted. Interesting is also that the good script works exceptionally well in the 100% twisted world. This is due to the fact that for this script many possible good twisted world exist.To summarize all results, twisting the world leads to less coverage of the world independently of whether basic actions or scripts are used. Furthermore, in the twisted world, it is less easy to clearly separate between “bad scripts” and “good scripts” because the twist can turn originally advantageous scripts into ineffective ones and vice versa.This paper contains three major points. First, the formalism for the creation of scripts from basic abilities to reduce the decision density; second, whether there exist scenarios in which scripts incur no additional control complexity for goal-directed navigation in both untwisted and twisted worlds; third, the merits of scripts for traversing the state space in general again comparing untwisted and twisted worlds. In all cases, we explored if and how scripts save resources for the agent and how the internal coding, the embodiment, of the basic actions impacts the cognitive cost.The core concept of a script is combining different basic actions to an action sequence accessible as a whole. Thus, the script is a composite action built out of basic capabilities to a new more complex capability. In reinforcement learning, the option framework comes to mind as achieving something similar: combining multiple actions to an option . Both apScripts are modeled after fixed action patterns which are highly specific to the animal . Many ofIn an untwisted world, adding scripts to the action space has shown to be informationally optimal for some setups but not for others. In For some setups, forcing the use of scripts by adding the decision cost to the reward increases the relevant information. In other words, the agent needs more precise knowledge (information processing) to take the decision what to do in a future step before it actually occurs, as seen in the results in With the introduction of the twisted world, we removed the well-ordered labeling of the basic actions and thus any specific external design regarding the information about global directions, pervading the whole world. The twists decouple the encoding of triggering actions (in our interpretation of the concept of “embodiment”) and their effect on the world and thus, removes the morphological computation aspect of the environment. We now consider how these automatic open-loop scripts interact with the environmental twists and under which circumstances a good twist can reduce the agent’s cognitive cost.In general, the results in Even though most twists make scripts difficult, some twists allow for the use of scripts. When the twists occur with a certain regularity, the scripts are actually able to represent twists within itself. Hence, scripts can embody this regular irregularity created by a particular twist along all paths to the goal see . FurtherFrom these results for goal-directed behavior in twisted worlds in Note here that the setups previously using scripts were not using the scripts anymore when the world was changed to a twisted one. Hence, to use scripts in an information-parsimonious context and thus, take fewer decisions, the embodiment of the agent and particularly its basic actions are important.In contrast to the previously discussed results, here the question was whether an agent reached a goal or how closely it could approach it without any information processing whatsoever. Next, we will discuss the affect of scripts and twists on the agent’s capability to traverse and cover the state space with limited information processing.west, north)  see  shows thDuring all experiments with a twisted world, the agent came close to fewer states than in an untwisted world. Hence, twists of the labeling of the basic action make traversing and covering the world harder, independently from the availability of scripts. In twisted worlds, the agent ended up visiting the same states over and over, creating the projected trajectories composed of an initial transient and an “attractor” the agent never leaves. Thus, we can say that a well-chosen labeling of basic actions plays a major role in the coverage of a state space with Zero-Information policies.This task cannot only evaluate an agent as a whole but also its individual actions, in other world evaluate a script. Using consistent labeling, scripts not traversing the state space are easier to spot. Since these scripts provide little value for navigation (only reaching two states in however many steps), these scripts can easily be identified as bad choices for the task, and thus adding a new sub-controller for it is a useless effort. Its potential relevance for modeling biological control mechanisms is that it is very important to know what not to spend resources on or into which skills not to invest. Hence, the information that a certain script is useless ahead of time is valuable information. However, in a twisted world, this decision is much harder. Almost every script has at least some merit somewhere in the world. In fact, the more the world is twisted, the more “traversing” all previously non-traversing scripts became. Hence, in an untwisted world non-traversing scripts are far easier to identify than in a twisted world.We studied relevant information in the framework of reinforcement learning and action scripts as well as untwisted (well-labeled) and twisted (ill-labeled) worlds. We were especially interested in the effect combining actions to scripts has on the relevant information. We further studied the importance of the embodiment of the basic action labels by comparing the untwisted world to varyingly twisted worlds.We defined a script, a fixed sequence of basic actions, as an extension to the reinforcement learning framework. A script creates new skills from the existing ones to composite actions, does not take feedback into account, is open-loop, and is similar to a fixed action pattern. Adding these to an agent, it now can decide on multiple future actions at once. However, adding all possible scripts creates a huge action space, and which scripts should be added to the action space, is an open question.We have analyzed the changes in information processing when using the new action space in goal-directed tasks We clearly see that there are cases for which the introduction of scripts allows the agent to save decisions without processing more information for an average decision. For other cases, we see this is not possible. This suggests that, depending on the task, scripts can save decisions at no additional cost.We then tested the scripts against a label-wise twisted world, that is, what we interpret in the abstract transition graph model of our world, as a different basic embodiment. Independently from the use of scripts or basic actions, the agent had to process far more information per decision. In most cases, the scripts would be used as little as possible. Overall, a high value of relevant information, whether because of the selected goal or the twistedness, makes the use of scripts less attractive. Interestingly, scripts have shown to work well with some special “ordered” twists in Limiting the agent to a Zero-Information policy while traversing the world, we clearly see scripts needing fewer decisions than basic actions. Still, trajectories generated by scripts are less accurate compared to basic actions. A twisted world is generally badly covered, independently of the use of scripts. This shows that a consistent labeling of basic actions which provides a kind of orientational semantics to the embodiment is important to save information processing during the tasks. This technique further allows us to evaluate an action with respect to its use for traversing the state space. Non-traversing scripts are easily spotted when the world is well-structured, a step toward reducing the action space to good scripts. In a twisted world, however, these “bad actions” are no longer as obviously bad and therefore harder to spot. Thus, a consistent labeling allows to identify more easily which scripts are useless to have in the repertoire. A well-adapted labeling of the basic actions allows one to make decisions more easily . This is further enhanced by the ability to create scripts which reduce the number of decisions overall.Among other, the results show that scripts are in general more sensitive to twists than basic actions. Why this is the case and what exactly causes it is a question for the future. We suspect that understanding this phenomenon will also give further insight into the cause when special twists as observed here occur which are then exploitable by scripts. In general, we see only some scripts being used at all. Thus, one goal of future research will be to predict such useful scripts , and only adding these to the action space and thereby reducing the huge action space that the current method requires.


Autologous fascial slings (AFS) have been used for a very long time in the treatment of female stress urinary incontinence, but the introduction of synthetic mesh slings placed either retropubicallyor trans-obturator has decreased the need to harvest the autologous rectus muscle fascia, thus reducing invasiveness and operative time. However AFS are still indicated in complicated cases and re-interventions, and the FDA has underlined safety concerns over the use of surgical meshes for the transvaginal repair of prolapsed pelvic organs.A 76-year-old woman with muscle-invasivebladder cancer underwent radical cystectomy 16 years after retropubic positioning of an autologous rectus muscle fascial sling for SUI, with complete symptom resolution. The sling was easily identified and removed en bloc with the bladder and urethra, providing an opportunity to histologicallyevaluate the autologous fascial graft after its long permanence in the new position. Histopathological examination demonstrated increased fibroblastic proliferation and formation of capillaries. A slight separation and an increased waviness of the connective fibers were both evident. An increased vascularity was also apparent, including transverse vessels, with clusters of vessels. A relative inflammatory reaction was present in over 300 cells/10 HPF. All these characteristics indicated viable connective tissue.AFS remain a valuable surgical option for both primary and recurrent SUI in women, showing high cure rates and low complications in the long-term. The present case, to the best of our knowledge, presents the longest follow-up period of an autologous rectus muscle fascia placed retropubically and its histological evaluation documents characteristics which support its mechanical strength and viability. Synthetic mid-urethral slings (MUS) have become the most frequently used surgical intervention for SUI for women The original AFS technique was first described over a century ago and consisted in the placement of a strip of rectus muscle fascia at the bladder neck. This approach is durable and effective but may lead to the appearance of voiding problems and de novo storage symptoms . A recenHowever,of the FDA has recently reviewed the literature and has underlined serious safety and effectiveness concernover the use of surgical mesh for the transvaginal repair of pelvic organ prolapse (POP). Patients treated with syntheticmeshes are subject to mesh related complications and sequelae (10% erosion within 12 months of surgery) and re-operation,and, moreover,the use of synthetic tissue does not appear to provide any added benefit when compared to traditional surgery . Within In November 2016 a76-year-old woman underwent radical cystectomy for a muscle invasive bladder cancer (MIBC) in our urology unit. The patient had a history of SUI afterhysterectomy and adnexectomy for an ovarian carcinoma in 1997. SUI was treated with an autologous fascial sling from the rectus muscle in 2000according to McGuire [The entire surgical specimen was fixed in 10% neutral-buffered formalin. The bladder neck, urethra and sling were isolated, embedded in paraffin and sectioned at 4–5 mm intervals, according to routine procedures. Histologic sections were stained with hematoxylin and eosin to evaluate fiber structure, the number of cells and vascularity. Increased fibroblastic proliferation was evident Fig. , and a mSince their introduction, syntheticmid-urethral slings have become the most commonly used surgical intervention for the female SUI, since they require shorter intraoperative and hospitalization times as well a shorter learning curve –5. A recMany studies analyzing the outcomes of synthetic mesh lack a long-term follow-up. A recent multicenter trial by Khan et al., however, found that even if there is not enough evidence to judge the long-term success rates between autologous and synthetic sling there is however some evidence that the “dry rate” for an autologous sling could be more durable. In the long term the AFS could be considered at least equivalent to TVT but with a higher success rate after mesh complication .The histological evaluation of the sample taken from our patient demonstrates the scarcity of studies availableon the characteristics of the material used in autologous slings in the long-term. The behavior over time of biological tissues used for reconstructive treatment could be the basis for the success or failure of the application of an autologous sling. Fitzgerald et al.  removed Animal models can provide useful information on how to integrate autologous tissues in the treatment of SUI or on which they can be most effective. McAdams et al. hypothesized that there may be a benefit in combining an autologous fascial graft and a deltoid flap into murine models .Autologous fascial graft, according to Carvalho et al. seems not to cause inflammatory reaction response in the histologic evaluation on grafted fascia in vocal fold of dog harvested two months from its implantation .The vitality of autologous grafted tissue many years after its implantation is not yet well known. Ahelen et al. hypothesized that there may be a regeneration and normalization, after a long period, of the semitendinosus tendon used for the reconstruction of the cruciate ligament. After an average of 8.4 years, they did not detect significant differences in the histological appearance between the regenerated tissues and those of the contralateral knee that had not been harvested .The greater awareness of the side effects related to the use of synthetic mesh has also made way for stem cells in the treatment of SUI or other diseases in which autologous tissues or synthetic implants are used. In a phase-one clinical study, Choi et al. evaluated the efficacy of a periurethral injection of adipose-derived autologous stem cells. The assessment was based on questionnaires related to continence but also on more objective methods  and evidenced better continence without indicating serious side effects . Orabi eWhat is interesting is the search for new surgical techniques which will be able to reduce the invasiveness of autologous slings. In a recent paper Osman et al. have illustrated a new andless invasive technique in which the autologous sling is positioned at the mid-level of the urethra and not at the level of the bladder neck. The midurethral placement of the graft is advantageous since it requires a lesser bladder neck dissection (thus avoiding the bladder base denervation and so lessening risk of detrusor overactivity and storage urinary symptoms), hematoma, bladder injury and fibrosis. This measure would allow patients with primary SUI to be treated with autologous material, thus extending the current indications which use the autologous sling mainly for patients with intrinsic sphincter deficiency or in the recurrences of SUI after a synthetic sling application. An additional benefit is that the learning curve of this technique is relatively short (approximately 20 procedures) .Woodruff et al. evaluated 24 grafts of different origin (polypropylene mesh(PPM), autologous fascia, porcine dermis, cadaveric dermis, cadaveric fascia) removed between 2 and 34 months after implantation. They demonstrated a more marked graft degradation in autologous and cadaveric fascia grafts. The PPM had the greater number of fibroblastic infiltrations but neovascularity was mostly represented in AFS . The hisThe renewed interest in autologous fascial slings in literature is supported both by its long-term efficacy on the symptoms and the reduced need for additional surgical adjustment and mesh-related compliances.The present case report aims to sustain this new trend of thought and to focus the attention on the autologous sling.In addition, an autologous fascial sling causes less fibrotic reaction than the synthetic, thus making future surgical treatments easier for SUI and other diseases.For commercial reasons the evolution of synthetic meshes has been extremely rapid and, since they are associated with a simpler implant technique, they have become the most frequently adopted surgical solution in the treatment of SUI. As a result, this has hindered the evolution and study of autologous tissues and their properties as possible grafts.The present histologic documentation of the preserved viability and strength of an autologous rectus fascia after being in use for 16 years provides further proof of the viability of this solution in the treatment of SIU.


The increase in ASM force induced by TNFα was not associated with an increase in regulatory myosin light chain (rMLC20) phosphorylation but was associated with an increase in contractile protein (actin and myosin) concentration and an enhancement of Ca2+ dependent actin polymerization. The sensitivity of ASM force generation to elevated [Ca2+]cyt (Ca2+ sensitivity) is dynamic involving both the shorter-term canonical calmodulin-myosin light chain kinase (MLCK) signaling cascade that regulates rMLC20 phosphorylation and cross-bridge recruitment as well as the longer-term regulation of actin polymerization that regulates contractile unit recruitment and actin tethering to the cortical cytoskeleton. In this study, we simultaneously measured [Ca2+]cyt and force responses to ACh and explored the impact of 24-h TNFα on the dynamic relationship between [Ca2+]cyt and force responses. The temporal delay between the onset of [Ca2+]cyt and force responses was not affected by TNFα. Similarly, the rates of rise of [Ca2+]cyt and force responses were not affected by TNFα. The absence of an impact of TNFα on the short delay relationships between [Ca2+]cyt and force was consistent with the absence of an effect of [Ca2+]cyt and force on rMLC20 phosphorylation. However, the integral of the phase-loop plot of [Ca2+]cyt and force increased with TNFα, consistent with an impact on actin polymerization and, contractile unit recruitment and actin tethering to the cortical cytoskeleton.Previously, we reported that in airway smooth muscle (ASM), the cytosolic Ca Previously, we and others showed that the regulation of [Ca2+]cyt in response to ACh stimulation is dynamic as reflected by small amplitude localized [Ca2+]cyt transients (Ca2+ sparks) that fuse into larger amplitude [Ca2+]cyt oscillations that propagate through the cell and summate into larger global responses  induce a cytosolic Caesponses . As ACh cine ASM . Furtheresponses .2+]cyt to a contractile response involves Ca2+ binding to calmodulin (CaM), Ca2+-CaM activating myosin light chain kinase (MLCK), which phosphorylates the regulatory myosin light chain (rMLC20) allowing cross-bridge recruitment and cycling  coupling of elevated cyt based on a calibration equation described by 2+]cyt and force responses of ASM strips to ACh concentration (EC50) were digitized (1 kHz sampling rate) using LabChart8 . The duplicate measurements were subsequently averaged for each trachea.Fura-2 fluorescence ion the ASM strip was excited using a mercury high-pressure lamp (75 W) as a light source at alternating (every 2 ms) wavelengths of 340 and 380 nm restricted by using a rotating filter wheel as previously described . Emitted20 phosphorylation was detected using a Phos-tagTM sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel with Zn2+ (20 phosphorylation. The second ASM strip in each group was stimulated with ACh concentration (EC50) for 30 s, a time period previously found to induce maximum rMLC20 phosphorylation  at the room temperature. The gel was immersed in 25 mM Tris, 192 mM Glycine, 10% methanol (v/v) containing 10 mM EDTA to remove Zn2+ for 30 min and proteins were transferred to polyvinylidene difluoride membrane, which was fixed with 0.5% formaldehyde in PBS for 45 min  and secondary antibody . Unphosphorylated rMLC20 and phosphorylated rMLC20 (p-rMLC20) were detected by enhanced chemiluminescence  and imaged on ChemiDoc MP Image System  and analyzed using an Image-Lab-Software   .n = 6) for the analysis of [Ca2+]cyt and force responses induced by ACh stimulation with (two strips) and without (two strips) 24-h exposure to TNFα. The two independent measurements of [Ca2+]cyt and force responses to ACh were then averaged to provide a single measurement per treatment per animal. In a separate set of four ASM strips from each animal (n = 6), baseline (no ACh stimulation) and ACh-induced changes in rMLC20 phosphorylation were analyzed with (two strips – one baseline and one ACh stimulated) and without (two strips – one baseline and one ACh stimulated) 24-h exposure to TNFα. Results were analyzed using a Student t-test . Data are summarized as means ± SD, with significance established at p < 0.05.Four ASM strips were used from each animal (n = 6), the duplicate measurements of ASM [Ca2+]cyt and force responses to ACh stimulation were comparable (<10% variance between the two measurements) and averaged to obtain a single value for summary analysis. In each ASM strip, ACh stimulation induced a biphasic [Ca2+]cyt response reaching an initial peak by ∼5 s and then declining to a sustained plateau after ∼2–3 min. The isometric force response to ACh stimulation was delayed and slower with maximum specific force reached after ∼1–2 min. Compared to control (untreated) ASM strips, 24-h exposure to TNFα (100 ng/ml) increased the peak force response to ACh by ∼18% compared to control (p < 0.05), whereas the peak [Ca2+]cyt response to ACh was unaffected by TNFα  ASM strips, 24-h TNFα exposure had no impact on the temporal delay between the onset of ACh-induced [Ca2+]cyt and force responses  (d[Ca2+]cyt/dt was ∼40 nM [Ca2+]cyt s–1. Compared to control (untreated) ASM strips, 24-h TNFα exposure had no significant effect on the rate of rise in ACh-induced [Ca2+]cyt response  . The maxresponse . The maxcontrols .2+]cyt and force responses in ASM strips was evaluated using phase-loop plots  ASM strips, TNFα significantly increased the integral of the phase-loop plot  induced by 30 s ACh stimulation was examined in ASM strips using western blot analysis  and enhanced cytoskeletal remodeling (actin polymerization) resulting in increased tethering of contractile elements to the plasma membrane and force translation to the extracellular matrix (ECM) rather than changes in rMLC20 phosphorylation -dependent inhibition of myosin light chain phosphatase (MLCP) , the force response in TNFα treated ASM was greater while the extent of rMLC20 phosphorylation was unaffected. In agreement, there was also no effect of TNFα exposure on the temporal delay between the onset of [Ca2+]cyt and force responses or on their rates of rise after ACh stimulation. These results indicate that neither the extent nor the rate of rMLC20 phosphorylation in ASM is affected by TNFα exposure. Thus, we conclude that ACh-dependent rMLC20 phosphorylation is not the primary mechanism through which Ca2+ sensitivity of force generation increases after 24-h TNFα exposure.It is commonly thought that an increase in Cae (MLCP) . Howeverereafter . This tincreases . Thus, w2+]cyt with a peak level reach in ∼5 s followed by a slow decline to steady state after ∼120–180 s. The initial elevation of [Ca2+]cyt in response to ACh stimulation is due to several sources of Ca2+ including inositol triphosphate receptor (IP3R) and ryanodine receptor (RyR) mediated sarcoplasmic reticulum (SR) Ca2+ release as well as extracellular Ca2+ influx via L-type, voltage-gated Ca2+ channels (3R and RyR channels are Ca2+ sensitive resulting in Ca2+-induced SR Ca2+ release (CICR) and a more rapid accelerating elevation of [Ca2+]cyt  reflect the short-term dynamic coupling mediated by rMLC20 phosphorylation and cross-bridge recruitment , we simultaneously measured force and ATP hydrolysis rate using an NADH-linked fluorescence technique  actin to filamentous (F) actin is Camulation . Zhang emulation . In suppmulation . Furthermulation , reflect2+]cyt and force responses evoked by electrical stimulation have been used to assess Ca2+ sensitivity of cardiomyocytes based on the [Ca2+]cyt at which 50% relaxation occurs (2+ binding to troponin-C (TnC) and thin filament regulation of cross-bridge attachment/detachment in cardiomyocytes. In contrast, the coupling of [Ca2+]cyt and force responses in ASM is far more complex; however, phase-loop plot of ACh-induced [Ca2+]cyt and force responses still provides valuable physiological insight. The rising phase of the phase-loop plot most likely reflects rMLC20 phosphorylation and cross-bridge recruitment, while the relaxation rate most likely reflects rMLC20 dephosphorylation. Importantly, both the rising phase and the relaxation rate of the phase-loop plots of ACh-induced [Ca2+]cyt and force responses were not affected by 24-h TNFα exposure. In contrast, the integral of the ACh-induced [Ca2+]cyt and force phase-loop plot increased significantly after 24-h TNFα.In the heart, phase-loop plots of [Can occurs . This as2+ sensitivity of ASM force generation through an effect on the long-term dynamic coupling of ACh-induced [Ca2+]cyt and force responses. Our results indicate that the increase in Ca2+ sensitivity induced by TNFα exposure is not dependent on rMLC20 phosphorylation or dephosphorylation. Instead, TNFα exposure increases actin and myosin concentration in ASM leading to the recruitment of additional contractile units. In addition, TNFα exposure increases actin tethering to cortical cytoskeleton and force translation to the ECM.We conclude that 24-h TNFα exposure increases the CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) Mayo Clinic.Y-SH, PD, and GS contributed the conception and design of the study, analyzed the data, performed the statistical analysis, and wrote the manuscript. Y-SH collected the data. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved.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.