Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these  In the vertebrate brain, immune cells including microglia can play both beneficial and detrimental roles in response to, for example, traumatic injury, stroke or neurodegenerative disease . A suppoMicroglia are self-renewing resident immune cells migrating into the brain in early embryonic development in vertebrates . InactivFollowing recruitment and/or activation of immune cells, phagocytosis is a key response to tissue damage, ensuring control of inflammation and tissue repair . In periin vivo live imaging studies on microglia performed in mice revealed that, unexpectedly, microglia under physiological conditions are highly dynamic  and peripheral immune cells such as monocyte-derived macrophages are activated to clear damaged and dying neurons. Clearance is followed by a termination phase, known as resolution of inflammation, in which immune cells that are no longer needed succumb to programmed cell death or exit the brain through lymphatic vessels. Currently, it is unclear which immune cells are involved at what stage of the disease process, and whether this response is beneficial or detrimental because macrophages and activated microglia are heterogeneous and a lack of selective markers has precluded the detailed study of their roles in conditions affecting the brain.Resultsin vivo. The authors use a previously established model to ablate brain cells in which transgenic nitroreductase enzyme causes cell death in targeted cells in the presence of a ligand. Using intravital imaging, they show that peripheral macrophages and resident microglia are both involved in clearing dying cells. In addition, they use electron microscopy to provide an unprecedented view of the cellular and ultrastructural features associated with neuronal ablation and the subsequent immune responses. Notably, they show that the timing of macrophage and microglia involvement is different, with macrophages being present at early stages whereas microglia dominate several days after the ablation. Other immune cells, including granulocytes, are not involved in the immune response. Finally, they show that after an initial increase, the numbers of immune cells in the brain decline, in part through phagocytes becoming apoptotic and being cleared by activated microglia.Here, the authors use intravital microscopy of cellular interactions in living zebrafish brain and electron microscopy to provide new insights into the immune response to brain injury, and to determine how neuroinflammation is stopped Implications and future directionsThese findings show that intravital microscopy in zebrafish can be used to discover immune maintenance mechanisms in the brain that would be missed using other approaches. Most surprisingly, they show that brain phagocytes undergo apoptosis and are engulfed by microglia, which suggests that inflammation in the brain can be resolved in a way similar to peripheral wounding responses, but that different immune cell types are involved. These novel findings could provide a handle for modifying the toxic, chronic neuroinflammatory processes that are found in many brain diseases.in vivo and how these are initiated and terminated. We find that dying neurons are initially effectively cleared by microglia and non-resident macrophages and subsequently by microglia, without involvement of infiltrating neutrophils or resident astrocytes. During the neuroinflammatory resolution phase, macrophage and microglia numbers decrease by exiting the central nervous system, and programmed cell death is followed by their phagocytosis by microglia.Our model provides a platform to study the dynamics of neuroinflammatory responses To address the nature and kinetics of leukocyte recruitment in response to cell death in the brain, we established a model system that allows controlled ablation of neurons: nitroreductase (NTR)-mediated cell killing in zebrafish larval brain  van Ham. TargeteA single overnight pulse of MTZ led to dose-dependent programmed cell death in 3-day-old fish larvae expressing neuronal NTR in the brain, but not in control zebrafish . Followisupplementary material Movie 1). In 5-day-old animals expressing neuro-NTR, MTZ also induced dose-dependent larval death . Relatively low doses of MTZ (2 mM) resulted in high levels of neuronal cell death and many fluorescently marked phagocytic cells not found in control larvae . However, over 80% of animals survived for over two weeks and grew to adulthood .To further define the roles of other immune cells, animals were studied later in development, when microglia and other immune cells have developed  . These results suggest that neuronal ablation is followed by a transient immune response that is not detrimental to recovery.To address how the immune response develops in such animals, phagocytes were counted on the basis of highly fluorescent phagosomes in the forebrains of animals at different stages after ablation. The fluorescent mCherry protein  is highlsupplementary material Table S1) . In addition to these morphological changes, the number of L-plastin-expressing cells in the forebrain region was increased twofold at 3 days after ablation  . In ablaable S1) . No debrablation .supplementary material Table S1). Granulocytes are normally not found in the brain but can infiltrate the neural tissue upon injection of bacteria into brain ventricles, or upon invasive spinal cord injury -driven GFP, resulting in GFP-positive granulocytes  . Complete sections of forebrains of control and NTR larvae treated with MTZ were subjected to EM . Cells tTo further classify the type of phagocytes in neuro-NTR animals treated with MTZ, large-scale electron microscopy (nanotomy) was performed, which allowed systematic large area analysis at nanoscale resolution . NanotomPrevious EM studies have identified macrophage-like and amoeboid microglia in vertebrates . Charactsupplementary material Movies 3–5). Upon imaging ApoE-GFP with higher laser power, we could detect the remaining phagocytic cell population, expressing low levels of GFP, which went unnoticed using conventional acquisition settings . Remarkably, some ramified microglia were present within 50 μm of regions with dying cells and phagocytic cells .Apolipoprotein E-driven membrane-tagged GFP (ApoE-GFP) clearly labels the microglia subset of mononuclear phagocytes, and ApoE-expressing microglia are the only known phagocytes in the brain after about 3 days of development . To elucsettings . This Apsupplementary material Movie 6), suggesting that some microglia exhibit a lack of (or a delayed) response to cell death compared with ApoE-low phagocytes. To address this possibility, the dynamics of the ratio of phagocytic microglia to ApoE-low phagocytes in the forebrain was determined. The numbers of ApoE-high microglia increased more than twofold between 1 and 3 days post-ablation (P≤0.005), whereas the numbers of ApoE-low phagocytes remained equal. The ratio of ApoE-high microglia to ApoE-low phagocytes increased from 57% at 1 day after ablation to 90% at 3 days after ablation . The increased proportion of microglia over time coincided with a relative decrease in ApoE-low phagocyte numbers, as numbers did not increase  . Additionally, phagocytic cell numbers in forebrains of NTR-ablated animals were increased more than twofold compared with controls at 1 day after ablation (Two types of mononuclear phagocytes have been characterized that express macrophage-expressed gene 1 (mpeg1)-GFP: peripheral macrophages and microglia . To addrable S1) . In contable S1)  and the able S1) . Furtherablation , in lineablation . Thus, aThe numbers of these phagocytes decline to basal levels 1 week after ablation . To examin vivo, NTR larvae treated with MTZ expressing mpeg1-GFP were imaged at this particular area, early after onset of neurodegeneration. The data suggest that migration along the olfactory nerve allows mpeg1-GFP macrophages to directly enter the brain . We conclude that two types of mpeg1-expressing mononuclear phagocytes are involved in clearance: microglial phagocytes showing high levels of ApoE-GFP and cells showing a very low level of ApoE-GFP expression. The latter might represent macrophages, which normally are not present in the brain and that could have entered the brain by migrating along the olfactory nerve .One mode of entry to the brain parenchyma for peripheral immune cells is by migration along the olfactory nerve fibers, through a porous bony plate called the cribriform plate, which separates the nasal cavity from the brain . To addrsupplementary material Movie 1). Analysis of a few remaining phagocytes with ingested debris 8 days after ablation revealed that some cells were no longer migrating and were immobilized, sometimes for several days . When we analyzed these cells in mpeg1-GFP expressing animals, we found that they showed apoptotic morphology and cytoplasmic GFP expression  and occasionally succeeding . Correlated microscopy . These features included vacuoles, amorphous membranous structures and lack of dense cytoplasm found in phagocytic cells; the latter being characterized by the presence of many organelles . Therefore, a decline in leukocyte numbers is achieved through phagocytes exiting the central nervous system and by programmed cell death of the phagocytes and their engulfment by microglia.Intravital microscopy revealed that microglia often appeared immediately next to these large phagocytic cell corpses, attempting to engulf the phagocyte  involved in clearance, suggesting that potentially phagocytic glial cells such as radial glia (the zebrafish counterparts of astrocytes) are not involved in engulfment and phagocytosis of cell debris in this model . We alsoA previous study on zebrafish microglia showed direct chemotaxis of microglia in response to localized acute laser-mediated cell death, and did not show involvement of other leukocyte classes . HoweverNo phagocytes except for ApoE-expressing microglia have been found in zebrafish brain at the stages we investigated under physiological circumstances . HoweverDespite a lack of obvious functional differences between the two cell types, they showed different temporal kinetics, with the macrophage-like cells mostly present in early stages, followed by microglia. Because some microglia are ramified and non-activated at those stages, macrophage-like cells might be more prone to respond in early stages, for example through a higher expression of receptors causing their recruitment. Simultaneously, there might be regional diversity of microglial phenotypes, and some brain areas might be more accessible to peripheral leukocytes . InteresOnly specific subclasses of leukocytes infiltrate the CNS under ablation conditions, suggesting that accessibility or recruitment of these cells in larval zebrafish brain to infiltration by immune cells is selective and regulated. Entry routes from the blood circulation in mammals could include migration across the wall of blood vessels in post-capillary venules or across the blood-cerebrospinal fluid barrier . AlthougThe phagocytic cells that remain in the CNS undergo programmed cell death and are sometimes engulfed by microglia. Although apoptotic microglia have been described in animal models as well as in human brain tissue, the fate of such cells is unclear. In peripheral wounding responses in vertebrates, recruited immune cells are generally short-lived and succumb to programmed cell death, a phenomenon known as resolution of inflammation . InfiltrZebrafish (mix of AB and longfins) were reared according to standard conditions . Embryosin vivo imaging , 1.5% potassium ferrocyanide (K4[Fe(CN)]6) on ice for 2 hours, and dehydrated in ethanol. Subsequently, larvae were incubated overnight in EPON 1:1 mixed with 100% ethanol, before embedding in EPON. EPON blocks were sectioned using an ultramicrotome . Semithin sections were stained using Toluidine Blue or Fuchsin as described. Ultrathin sections were mounted on Formvar-coated nickel grids and stained with lead citrate and uranyl acetate. Transmission electron microscopy (TEM) images were recorded using a FEI CM100 operating at 80 kV equipped with a Morada digital camera . For large-scale EM, acquisition was different to that previously described when we used camera-based recording using TEM  was used as described . For multicolor two-photon microscopy of red (mCherry) and green (GFP) fluorophores the laser  was tuned to ~970 nm (940–970 nm) and 500–550 and 575–610 filter sets were used. A 20× dipping objective  was used for these experiments. Multicolor confocal microscopy was carried out on a LSM780 system equipped with a transmission detector for DIC using Zen software. PlnApo 20×, 0.8 NA DICII and Lcl Plan Neofluar 63×, 1.3 NA lenses were used and 405, 488, 514, 561, 594 and 633 laser lines. An automated stage was used on both 7 MP and LSM780 systems to acquire multiple time-lapse recordings simultaneously, and acquire escribed . For 3D NTR zebrafish treated with MTZ and control zebrafish were allowed to recover up for several weeks. To dissect their brains, animals were first anesthetized on ice, and heads were severed behind the gills. The lower jaw, gills and eyes were removed using watchmaker’s forceps and brains were fixed inside the skull in Zamboni fixative  overnight at 4°C. Skulls containing whole brains were rinsed twice in 0.1 M sodium cacodylate and dissected from the skull using forceps  and a 20-gauge hypodermic needle. Images of fixed zebrafish brains were acquired using a Leica fluorescence dissection microscope. Subsequently, osmium fixation and embedding in EPON of whole brains was performed as described for electron microscopy. Sections were stained using Toluidine Blue and basic Fuchsine and visualized using an Olympus microscope.Zebrafish were treated with MTZ as described . BrieflyTransgenic lines used in this study were combinations between MPX-GFP  and MPEGN-phenylthiourea , low melting point (LMP) agarose (Sigma).Metronidazole (Sigma), tricaine methanesulfonate (MS222) (Sigma), t-test. For image processing and quantitative analysis, Excel (Microsoft), Photoshop (Adobe), Prism (Graphpad), Fiji ImageJ, Zen (Zeiss) and Imaris (Bitplane) were used.Statistical significance was calculated using the Student’s


Indy (I’m Not Dead Yet) encodes the fly homolog of a mammalian SLC13A5 plasma membrane transporter. INDY is expressed in metabolically active tissues functioning as a transporter of Krebs cycle intermediates with the highest affinity for citrate. Decreased expression of the Indy gene extends longevity in Drosophila and C. elegans. Reduction of INDY or its respective homologs in C. elegans and mice induces metabolic and physiological changes similar to those observed in calorie restriction. It is thought that these physiological changes are due to altered levels of cytoplasmic citrate, which directly impacts Krebs cycle energy production as a result of shifts in substrate availability. Citrate cleavage is a key event during lipid and glucose metabolism; thus, reduction of citrate due to Indy reduction alters these processes. With regards to mammals, mice with reduced –/–)Indy (mIndy also exhibit changes in glucose metabolism, mitochondrial biogenesis and are protected from the negative effects of a high calorie diet. Together, these data support a role for Indy as a metabolic regulator, which suggests INDY as a therapeutic target for treatment of diet and age-related disorders such as Type II Diabetes and obesity. Drosophila I’m Not Dead Yet (Indy) gene encodes a plasma membrane transporter of Krebs cycle intermediates with highest affinity for citrate , which is an environmental manipulation that extends longevity in a variety of species  show the highest levels of expression in the liver and brain  has the highest sequence and functional similarity to Drosophila Indy. There is 34% identity and 50% similarity between the predicted INDY protein in flies and the human and rat sodium dicarboxylate transporter. In flies INDY is mainly expressed in the midgut, fat body and oenocytes  dilp2, dilp3, and dilp5 when kept on high caloric diet (HCD), with levels similar to genetic control flies on a CR diet , which transports citrate from the mitochondria thereby contributing to cytoplasmic citrate regulation .spargel/dPGC-1, increases mitochondrial biogenesis to maintain cell energetic requirements (Figure PGC-1 mRNA levels and mitochondrial biogenesis in –/–mIndy mice and the midgut of flies with reduced INDY levels (–/–mIndy mice have higher lipid oxidation, reduced lipogenesis and increased insulin sensitivity (Reduced INDY activity in flies and mice alters availability of these substrates during intermediary metabolic processes by reducing citrate transport, subsequently causing reduction of ATP levels . Augments Figure . This isY levels . Consistsitivity .Indy mRNA in the fly midgut, while CR had the opposite effect (Indy gene expression has recently been provided showing altered levels of mIndy gene expression in rat hepatocytes following glucagon release during early starvation (mIndy (Indy mRNA expression, while prolonged starvation decreases expression levels. This is most likely due to the short half-life of glucagon and the subsequent activation of downstream regulatory feedback loops (Indy mRNA found in the livers of mice after 36 h of starvation. Furthermore, increased INDY expression was found in the rat liver after force-feeding large amounts of olive oil, thus supporting a regulatory role for INDY during metabolic activity (Environmental manipulations can mimic aging, such as exposure to HCD or paraquat. It was recently shown that such manipulations also increase levels of e effect . Given tarvation . Glucagon (mIndy . Overnigck loops . These factivity .Aging is a complex process characterized by a loss of homeostasis . PreservIndy levels are associated with dramatic extension of lifespan accompanied by enhanced metabolic activity. We have shown that reduction in Indy mediates changes in intermediary metabolism that preserves ISC regenerative homeostasis and intestinal integrity by modulating dPGC-1 activity (Indy mRNA levels have significantly higher dPGC-1 mRNA levels in the midgut throughout lifespan compared to genetic controls, which exhibit an age-related reduction in the levels of dPGC-1. Not only do these conditions promote oxidative stress resistance, but also preserve the redox environment of ISCs, which subsequently preserves proliferative homeostasis and intestinal integrity. Without functional dPGC-1, Indy mutant flies do not show beneficial changes in mitochondrial physiology, ROS production or intestinal integrity, suggesting that dPGC-1 mediates some of the regulatory effects of Indy-mediated healthspan.Recently we described a role for INDY in the midgut during intestinal regeneration. INDY is robustly expressed in midgut tissue. Reduce activity . Flies wdPGC-1 and Indy also has a large impact on Drosophila longevity. We showed that Indy and dPGC-1 longevity pathways overlap, which was supported by the observation that flies hypomorphic for Indy and dPGC-1 have a lifespan similar to controls (dPGC-1 must be present and functional in order to confer lifespan extension in Indy alleles. This is consistent with recent work that has revealed a role for the dPGC-1 in ISC homeostasis, establishing a connection between mitochondrial function and tissue homeostasis (dPGC-1 activity (Indy increases dPGC-1 activity, which promotes mitochondrial biogenesis and changes the redox environment of the Indy mutant midgut. Such changes preserve tissue homeostasis and ultimately mediate lifespan extension.The relationship between controls . Such fieostasis . Our woractivity . We suggIndy gene activity is comparable to CR and associated with a longer and healthier life by affecting energy production. Similarly, knockdown of –/–mIndy in mice mimics CR by altering metabolic activity in the liver (Longevity studies in worms and flies demonstrated that reduction in he liver . The reche liver . The simThe 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.


In our studies, these porous membranes significantly improved nerve fiber regeneration and functional recovery in rat models of axonotmetic and neurotmetic sciatic nerve injuries. In particular, the number of regenerated myelinated nerve fibers and myelin thickness were significantly higher in rat treated with chitosan porous hybrid membranes, whether or not they were used in combination with mesenchymal stem cells isolated from the Wharton's jelly of the umbilical cord. In this review, we describe our findings on the use of chitosan-GPTMS hybrids for nerve regeneration.The treatment of peripheral nerve injuries remains one of the greatest challenges of neurosurgery, as functional recover is rarely satisfactory in these patients. Recently, biodegradable nerve guides have shown great potential for enhancing nerve regeneration. A major advantage of these nerve guides is that no foreign material remains after the device has fulfilled its task, which spares a second surgical intervention. Recently, we studied peripheral nerve regeneration using chitosan- Nerve regeneration is a complex biological process. While approaches for peripheral nerve repair have improved over the last few decades, functional recovery is usually incomplete. As a result, much attention has been given by researchers and clinicians to cell-based therapies and tissue engineering –4. AutogChitosan and its complexes have been studied for a number of biomedical applications, including wound dressings, drug delivery systems, and space-filling implants, because of their biocompatibility, biodegradability, wound-healing, and antibacterial properties . Nerve cChitosan nerve tube guides have also been modified by some research groups with inorganic components. For example, Gärtner et al. prepared chitosan hollow tubes from crab tendons  and modiWallerian degeneration is the process of degeneration of the axon distal to the site of transection. Once a peripheral nerve has been transected, Wallerian degeneration of the distal axons begins and macrophages enter the area to remove the myelin and axonal debris. The surrounding basement membrane and Schwann cells play important roles in this process. Schwann cells line up along the basement membrane tube and synthesize growth factors that attract axonal sprouts formed at the terminal of the proximal segment of the severed axon . Adding  in vitro. Because NSCs are multipotent stem cells that can follow multiple differentiation pathways, using this differentiated cellular system may allow resident cell replacement and create an environment that supports and improves axon regeneration. The authors found that 50 ppm of gold nanoparticles stimulated cell proliferation and gene expression. After 6 weeks of implantation, a better and faster functional recovery was observed in animals treated with the chitosan-gold nanocomposite and NSCs compared with the composite alone in their model of neurotmetic injury with a gap. Histomorphometric analysis revealed that the number of myelinated axons in the regenerated nerve fibers was higher in animals where the nerve was reconstructed with the chitosan-gold nanocomposite and NSCs [Yamaguchi et al. , 34 modiand NSCs .γ-glycidoxypropyltrimethoxysilane (GPTMS) units using sol-gel methods [29Si and 13C CP-MAS NMR analysis, FT-IR spectroscopy, ninhydrin assay, and contact angle analysis revealed the following physical characteristics of these hybrids [Our group has synthesized inorganic-organic hybrids derived from chitosan and  methods –42. Diff hybrids , 38: (1)μm and a very high porosity, normally more than 85% [The solid chitosan hybrid membranes allowed for good proliferation and differentiation of the human osteoblastic cell line MG63 and harvested human bone marrow cells. The attachment, proliferation, and alkaline phosphatase (ALP) activity of MG63 cells were higher when cultured on chitosan hybrid membranes compared with chitosan membranes , 38. Humthan 85% . The celthan 85% .2 exchange [Amado et al. studied nerve regeneration using solid and porous chitosan-GPTMS membranes in a standardized crush or axonotmetic injury of the rat sciatic nerve . The 3 mexchange . These pexchange –51.Simões et al. tested the histocompatibility of the biomaterials developed for nerve regeneration by implanting the solid and porous membranes subcutaneously in the rat model. These membranes were subsequently used in experiments on sciatic nerve regeneration after axonotmetic and neurotmetic injuries . The porSimões et al. investigated the effect of the porous membranes in surgical neurotmetic repair either by direct suture, autograft, or tubulization and also compared a tube guide made of poly(lactide-co-glycolic) acid, where the two monomers, the lactic acid and the glycolic acid, were in a ratio of 90 : 10 (PLGA 90 : 10) . As show in vitro differentiated N1E-115 cell line [ In vivo experiments using the rat sciatic nerve model were performed using the porous hybrid chitosan membranes seeded with neuroglial-like cells obtained from in vitro differentiated N1E-115 cells. The combination of the porous hybrid membranes with these neuroglial-like cells was tested in axonotmetic and neurotmetic injuries. The implantation of cultured cells  into defective nerves can be achieved using two different techniques. The cellular system can be directly injected into the neural scaffold that has been interposed between the proximal and distal nerve stumps or around the crush injury . Alternatively, implantation can be achieved by preadding the cells to the neural scaffold via injection or coculture , and then the biomaterial with the cellular system is implanted into the injured nerve [ in vitro cultured model of stem cells [Schwann cells, mesenchymal stem cells, embryonic stem cells, and marrow stromal cells have been extensively studied for their ability to promote nerve regeneration. We focused our research on theell line –51 and oell line . In vivoed nerve –51. The ed nerve . The celem cells .Simões et al., in 2010, used the rat sciatic nerve model for investigating the effect of porous hybrid chitosan membranes in a neurotmetic injury, used together with standard microsurgical repair methods  . Morphol in vitro differentiated N1E-115 cells suggested that the impairment in nerve fiber regeneration in these systems might have been caused by the presence of a large number of neuroblastoma-like cells colonizing large areas of the nerve profile, which might have interfered with nerve regeneration inside the nerve guides. Interestingly, the presence of many blood vessels was observed inside the large neuroblastoma-like clusters, suggesting that transplanted cells can deprive the regenerating nerve fibers of blood supply [ in vitro differentiated into neuroglial-like cells, did not facilitate either nerve regeneration or functional recovery in neurotmetic injuries when used in combination with PLGA 90 : 10 or porous chitosan membranes. Indeed the presence of this cellular system reduced the number and size of the regenerated fibers, thus suggesting that this type of cellular system can partially impair nerve regeneration, at least from a morphological point of view. The impaired axonal regeneration seems to be the result of N1E-115 cells surrounding and invading the regenerating nerve, since many of these cells were seen colonizing the nerve and might have deprived the regenerating nerve fibers of blood supply. Taken together, these results suggested that the N1E-115 cells do not promote nerve healing, and their use might even impair nerve regeneration. For this reason, we started using human MSCs [Light and electron microscopy analysis of PLGA 90 : 10 and chitosan tube guides containingd supply , 50. It man MSCs , 50.Gärtner et al. seeded the porous membranes with undifferentiated human MSCs (hMSCs) isolated from Wharton's jelly . MSCs isIn vivo studies indicate that chitosan-GPTMS porous hybrid membranes are a very promising clinical tool in peripheral nerve reconstructive surgery. The combination of the chitosan-GPTMS porous hybrid membranes and hMSCs has a slight advantage in comparison to untreated controls. An enhancement of nerve regeneration was observed when hMSCs or the porous hybrid chitosan membranes were used alone but not when used in combination. An increase in myelin production, visible as higher myelin thickness measured by histomorphometry, was observed in surgically reconstructed nerves after axonotmetic and neurotmetic injury. These findings demonstrate the therapeutic potential of these cells and biomembranes in neurodegenerative diseases that are typified by demyelination. Further investigation using animal models is required to determine the efficacy of these systems in the treatment of critical nerve defects and neurodegenerative diseases.


Music-induced brain activity modulations in areas involved in emotion regulation may be useful in achieving therapeutic outcomes. Clinical applications of music may involve prolonged or repeated exposures to music. However, the variability of the observed brain activity patterns in repeated exposures to music is not well understood. We hypothesized that multiple exposures to the same music would elicit more consistent activity patterns than exposure to different music. In this study, the temporal and spatial variability of cerebral prefrontal hemodynamic response was investigated across multiple exposures to self-selected musical excerpts in 10 healthy adults. The hemodynamic changes were measured using prefrontal cortex near infrared spectroscopy and represented by instantaneous phase values. Based on spatial and temporal characteristics of these observed hemodynamic changes, we defined a consistency index to represent variability across these domains. The consistency index across repeated exposures to the same piece of music was compared to the consistency index corresponding to prefrontal activity from randomly matched non-identical musical excerpts. Consistency indexes were significantly different for identical versus non-identical musical excerpts when comparing a subset of repetitions. When all four exposures were compared, no significant difference was observed between the consistency indexes of randomly matched non-identical musical excerpts and the consistency index corresponding to repetitions of the same musical excerpts. This observation suggests the existence of only partial consistency between repeated exposures to the same musical excerpt, which may stem from the role of the prefrontal cortex in regulating other cognitive and emotional processes. Music is capable of modulating brain activity in regions involved in emotional processing  , has motivated the use of music as an intervention for individuals experiencing anxiety or depression symptoms –6. In a 2] and [Hb]) and is suited for long-term bedside monitoring in adults, children, and neonates. These characteristics render NIRS plausible for naturalistic studies of emotion-related brain activity. Recent findings have confirmed significant associations between prefrontal hemodynamic response, measured using NIRS, and emotional experience [2]. Similarly, Tai and Chau were able to differentiate the response to affective pictures from baseline with an average of 75% accuracy, using prefrontal cortex NIRS  and [Hb] by applying the modified Beer–Lambert law [2], using the method introduced by Cui et al. [2].An Imagent Functional Brain Imaging System from ISS Inc.  was use for NIRS measurements across nine different regions on the forehead, as shown in bert law . The [Hbi et al. . Further2] were compared by using instantaneous phase. For each recording region shown in The hemodynamic changes were compared across the four repetitions by introducing a spatio-temporal measure of consistency. The spatial i.e., regional, patterns of [HbO1, L4, R1, and R4 were included for the comparison, and for L3, regions L2 and L4 were included for comparison. The comparison was conducted by finding the mean squared error between the neighboring regions (within 3 cm radius) and each recording location. Hence, for the previous example corresponding to L3, the mean squared error (MSEL3) was determined as shown in  value. Therefore, MSEregion(t) was used as a measure of instantaneous phase distinction for each recording region . To emphasize larger distinctions, the MSE was exponentially mapped to the phase distinction waveform (PDW) as shown in  from each other.In the previous phase, the It is important to note that the two analysis methods i.e., consistency index and pair-wise consistency index are proposed to address two different questions. The consistency index compares all four repetitions and the pair-wise consistency index assesses similarity among a subset of trials.P vector was estimated for all 50 iterations of randomly matched experimental blocks. For each iteration the P vector for the randomly matched blocks was compared to P vectors corresponding to the experimental blocks. It is important to note that the four musical excerpts in the same group were randomly selected non-identical excerpts. A Kolmogorov-Smirnov test was carried out to assess the significance of any differences observed (p<0.05).The A list of the songs selected by participants is presented in e and f (corresponding to comparisons between 2nd and 4rd repetitions and 3rd and 4th repetitions) are very similar but differ substantially from b. Such observations showed the importance of investigating consistency between subsets of the repeated music pieces in addition to all four repetitions.The similarity topographies for participant 1 are shown in consistency index corresponding to repeated musical excerpts and randomly matched sets of non-identical musical excerpts did not reach significance (p<0.05) for any of the 50 iterations. Therefore, no significant difference was observed in the spatiotemporal consistency of the identical self-selected songs and the consistency among exposures to non-identical musical excerpts (i.e. randomly matched sets) when comparing all four repetitions.The difference between the pair-wise consistency indexes (representing similarity topographies). The resulting P vectors (pair-wise consistency indexes were significantly (p<0.05) different for identical musical excerpts compared to non-identical (randomly grouped) musical excerpts. For pair-wise consistency indexes that were significantly different, identical musical excerpts resulted in larger consistency indexes. This result suggested that when comparing smaller subsets, repeated exposure may result in more consistent spatio-temporal patterns.In the second phase, illustrated in The distribution of emotional ratings varied across individuals and for various self-selected musical excerpts. Therefore, the emotions experienced during different exposures to the same musical excerpt were not similar. The distribution of the reported ratings of arousal and valence are depicted in boxplots shown in consistency index was introduced. No significant differences were identified in the hemodynamic response variability (measured using consistency indexes) during 4 repetitions of the same musical excerpts, compared to hemodynamic response variability during different musical excerpts. However, the hemodynamic response variability (measured using consistency indexes) were significantly different for identical versus non-identical musical excerpts when comparing a subset of repetitions. Therefore, the current results suggest only partial consistency in the observed prefrontal hemodynamic response.The current results were obtained using near-infrared spectroscopy (NIRS) which is suited for prolonged monitoring of prefrontal cortex hemodynamics in naturalistic studies. NIRS is non-invasive, relatively inexpensive, and portable. Therefore, NIRS, which is frequently used in clinical settings for monitoring neonates, children and adults, may be useful in investigating the effect of music as a clinical intervention for these populations. In prolonged studies involving music, repeated exposure to the same musical excerpts may be required. For designing such studies, it is necessary to understand the variability in the prefrontal cortex hemodynamics during repeated exposure to the same musical excerpt, particularly due to the potential influence of music familiarity –11. To mWhen comparing all four repetitions, the similarity indexes were not significantly different from randomly matched non-identical musical excerpts. However, there was a significant difference between the pair-wise similarity indexes in identical versus randomly-matched non-identical pairs of musical excerpts. This observation indicates that although all repetitions did not result in similar hemodynamic activity patterns, similarities were observed among subsets of these repetitions which were significantly different from randomly matched non-identical musical excerpts.Previous studies have identified networks specialized for perceiving specific music characteristics –25. ThesRepeated exposures to the same musical excerpt, which shared identical musical characteristics, resulted in different activity patterns in many cases  as a marker of musical syntax processing and found increased ERAN amplitude in musicians compared to non-musicians . In anotFunctional NIRS (fNIRS) experiments with our prefrontal set- up does not provide neuroanatomical resolution specificity of brain fMRI, however, it could allow for recordings that could be extended for hours and many days . The light sources and detectors were held in place using a custom-made non-elastic headset which was centered at midline and above the eyebrows in each trial to ensure consistency. However, no information was acquired regarding the underlying neuroanatomical structures. Due to this limitation, we refrained from making any conclusions based on the underlying anatomical structures. Future investigations should consider using a structural imaging modality such as MRI in combination with NIRS to determine the exact neuroanatomical distribution of the observed hemodynamic changes.In this study, prefrontal hemodynamic response during repeated exposure to identical musical excerpts was investigated using NIRS technology. For this purpose, a spatiotemporal measure of consistency was introduced. The consistency among four repeated exposures to the same musical excerpt was not significantly different from consistency among non-identical randomly matched musical excerpts, while pair-wise comparisons revealed consistencies which were significantly different from non-identical musical excerpts.These results highlight the importance of emotional and cognitive processes that may engage prefrontal cortex during different occasions of exposure to identical musical excerpts. Finally, future studies involving dynamic emotional monitoring in addition to prefrontal hemodynamic recording are warranted.


Current upper limb prostheses do not replace the active degrees-of-freedom distal to the elbow inherent to intact physiology. Limited evidence suggests that transradial prosthesis users demonstrate shoulder and trunk movements to compensate for these missing volitional degrees-of-freedom. The purpose of this study was to enhance understanding of the effects of prosthesis use on motor performance by comparing the movement quality of upper body kinematics between transradial prosthesis users and able-bodied controls when executing goal-oriented tasks that reflect activities of daily living.Upper body kinematics were collected on six able-bodied controls and seven myoelectric transradial prosthesis users during execution of goal-oriented tasks. Range-of-motion, absolute kinematic variability (standard deviation), and kinematic repeatability (adjusted coefficient-of-multiple-determination) were quantified for trunk motion in three planes, shoulder flexion/extension, shoulder ab/adduction, and elbow flexion/extension across five trials per task. Linear mixed models analysis assessed between-group differences and correlation analysis evaluated association between prosthesis experience and kinematic repeatability.Across tasks, prosthesis users demonstrated increased trunk motion in all three planes and shoulder abduction compared to controls (p ≤ 0.004). Absolute kinematic variability was greater for prosthesis users for all degrees-of-freedom irrespective of task, but was significant only for degrees-of-freedom that demonstrated increased range-of-motion (p ≤ 0.003). For degrees-of-freedom that did not display increased absolute variability for prosthesis users, able-bodied kinematics were characterized by significantly greater repeatability (p ≤ 0.015). Prosthesis experience had a strong positive relationship with average kinematic repeatability .The use of shoulder and trunk movements by prosthesis users as compensatory motions to execute goal-oriented tasks demonstrates the flexibility and adaptability of the motor system. Increased variability in movement suggests that prosthesis users do not converge on a defined motor strategy to the same degree as able-bodied individuals. Kinematic repeatability may increase with prosthesis experience, or encourage continued device use, and future work is warranted to explore these relationships. As compensatory dynamics may be necessary to improve functionality of transradial prostheses, users may benefit from dedicated training that encourages optimization of these dynamics to facilitate execution of daily living activity, and fosters adaptable but reliable motor strategies.The online version of this article (doi:10.1186/1743-0003-11-132) contains supplementary material, which is available to authorized users. The musculoskeletal architecture of the upper body  contains redundant degrees-of-freedom (DoFs) from which the central nervous system (CNS) has the benefit of selecting various task-equivalent motor strategies and associated inter-joint coordination patterns to satisfy task requirements –3. The aDespite indication that compensatory motions may result in overuse injuries , 14, theThe objectives of this study were to compare upper body movements and their associated variability between able-bodied individuals and transradial prosthesis users during execution of goal-oriented tasks that reflect activities of daily living (ADLs), and to assess relationships between movement variability and prosthesis experience. We hypothesize that: 1) prosthesis users will display altered range-of-motion (RoM) at the trunk and shoulder compared to able-bodied individuals to compensate for a loss in active distal DoFs, 2) prosthesis users will display increased within-subject movement variability as they recruit compensatory motions to execute novel goal-oriented tasks, and 3) movement repeatability of prosthesis users has a positive direct relationship with prosthesis experience. Importantly, upper limb prosthesis use performance has traditionally been assessed through the use of clinical outcome measures –30. AlthParticipant inclusion criteria required that they be free of neurological and musculoskeletal pathology (apart from upper limb loss) that would impair upper limb motor control during goal-oriented task execution in a seated position. Participants with limb loss were required to have unilateral loss at the transradial level and currently operate a prosthesis with myoelectric control that would be used during the experiments. A requirement was that the prosthesis be self-suspending with a supracondylar socket design. Ethical approval was obtained from the University Institutional Review Board, and informed consent was provided by each participant prior to data collection.Six able-bodied individuals  and seven individuals with limb loss at the transradial level  participated in this study. Limb loss etiology included traumatic (n = 4) and congenital (n = 3). User devices included the System Electric Hand , MyoHand VariPlus Speed Hand , Transcarpal Hand , Motion Control Hand , i-Limb Ultra Revolution , i-Limb Ultra , and i-Limb Hand . For all participants with traumatic amputation, the amputated limb was considered their dominant limb prior to the amputation and the average time since amputation was 9.5 ± 11.0 years. Across all subjects, the average reported prosthesis experience was 20.3 ± 18.1 years. Prosthetic socket comfort was quantified with the socket comfort score , which aFood cutting – picking up and positioning a knife to slice through a small plasticine roll located at midline of the body.Page turning – grasping a page, located on one side of the body, with the hand on the contralateral side and turning the page over to the ipsilateral side as if turning the page of a book.Carton pouring – lifting a carton, located at midline of the body, and emptying the liquid contents into a jar on the contralateral side with minimal spilling.Lifting and transferring a weighted object – lifting a liquid-filled jar, located on one side of the body, with the hand on the contralateral side and transferring to the ipsilateral side over a low-level barrier at midline of the body.Lifting and transferring a tray – lifting a tray, located on the contralateral side of the body, with both hands and transferring to the ipsilateral side over a low-level barrier at midline of the body.Participants were requested to execute five goal-oriented tasks while seated at a table of standard height . The tasks and associated protocol were adopted from the Southampton Hand Assessment Procedure (SHAP) , as thisPer the SHAP protocol, participants were requested to complete each task as fast as possible. Tasks did not require participants to position their joints/segments in any particular orientation in space, but only to complete the task requirements. Able-bodied controls and prosthesis users performed these tasks using their non-dominant and prosthetic limb, respectively, for unimanual tasks . For the tasks of page turning and lifting and transferring a weighted object and tray, the object was first positioned on the side of the body opposite to the non-dominant or prosthetic limb. Prior to data collection and the start of each task, the participants rested both hands on the table in the same position Figure a. Once tKinematic data were collected with a digital motion capture system  at 120 Hz using a custom upper extremity retro-reflective marker set to define three rigid-body segments  and three additional markers on the table surface to define the work plane. A subset of the anatomical locations of retro-reflective markers used in this analysis include: C7 spinous process, sacrum, top of the sternum, bilateral acromion processes, medial and lateral humeral epicondyles, and radial and ulnar styloids. For prosthesis users, the locations of the epicondyle and styloid markers mimicked the relative position of these bony landmarks on the intact arm. Virtual elbow and wrist joint centers were created as the midpoint between the epicondyle and styloid markers, respectively. The forearm was defined as the segment linking the virtual wrist and elbow centers, and the upper arm was defined as the segment linking the virtual elbow center and acromion process marker.Upper body kinematics  were estimated from the five trials of each task using custom software . A trunk-based local reference frame was calculated using the C7, sacrum, and sternum markers to define the anatomical sagittal, frontal, and transverse planes. Shoulder flexion/extension was defined as the angle between the upper arm and trunk anterior-posterior axis . Shoulder abduction was defined as the angle between the upper arm and shoulder axis created by the acromion markers . Elbow flexion/extension was defined as the angle between the upper arm and forearm (180° of angular displacement was defined as full elbow extension). Trunk lateral flexion was defined as the angle between the trunk inferior-superior axis and table medial-lateral axis  projected onto the frontal plane . Trunk transverse rotation was defined as the angle between the shoulder axis and table medial-lateral axis projected onto the transverse plane . Trunk forward flexion was defined as the angle between the trunk inferior-superior axis and table anterior-posterior axis projected onto the sagittal plane . Kinematic data were filtered with a bidirectional 4th order low-pass Butterworth filter at 6 Hz. DoF angle trajectories were normalized to task completion time .R2 value [For each task, average RoM, absolute kinematic variability and kinematic repeatability of the observed DoF angles across five trials were calculated for each participant. The RoM of each DoF was estimated by subtracting the maximum and minimum recorded angles. Absolute kinematic variability across the five trials was estimated as the average standard deviation (SD) across all time points of the normalized DoF angle trajectory. Kinematic repeatability was estimated by the adjusted coefficient of multiple determination (CMD), also commonly reported as the R2 value . The CMDR2 value , with vaR2 value , 35. ConR2 value , 35. In R2 value –41.Statistical analyses were conducted using SPSS  and the critical alpha was set at 0.05. Normality of the data was confirmed using the Shapiro-Wilk test. A linear mixed model (LMM) analysis  was used to assess if group classification resulted in individual DoF RoM, SD, and CMD differences. Group means and 95% confidence intervals were calculated for each task to illustrate task-specific differences and group × task interaction effects. An overall CMD value was calculated for each participant by averaging CMD values across DoFs and tasks  and a Pearson correlation coefficient was estimated between prosthesis users’ overall CMD values and prosthesis experience.The majority of prosthesis users were unable to routinely execute tasks 1 (food cutting) and 2 (page turning) as required by the protocol due to their inability to maintain grip of the knife and fully rotate the page from front to back, respectively, with their prostheses. Consequently, data from these tasks were eliminated from the analysis. Additionally, one prosthesis user was unable to execute task 5 (tray lift and transfer) due to inadvertent opening of the prosthetic hand and these data are missing from the analysis. To aid in visualization of kinematic results, an example of group ensemble average kinematics of task 3 (carton pouring) is presented in Figure Results indicate that prosthesis users demonstrate a significant increase in shoulder abduction, trunk transverse rotation, trunk lateral flexion, and trunk forward flexion RoM. Additionally, for prosthesis users, all of the DoFs with increased RoM apart from trunk forward flexion also displayed increased absolute kinematic variability as reflected by increased average SD. For shoulder flexion/extension, elbow flexion/extension, and trunk forward flexion, which did not display differences in absolute kinematic variability (SD), able-bodied controls demonstrated increased kinematic repeatability as reflected by greater CMD values. Few interaction effects (group × task) were revealed by the statistical analyses, and these were primarily limited to RoM of shoulder flexion/extension and abduction, and trunk lateral and forward flexion  the loss of active distal DoFs associated with a transradial prosthesis encouraged alterations in kinematic RoM, 2) transradial prosthesis users demonstrated increased kinematic variability, and 3) prosthesis experience was associated with increased kinematic repeatability.Prosthesis users demonstrated greater shoulder abduction and trunk RoM than the able-bodied controls when observed across tasks Figure . GeneralOverall, these results seem to support the idea that it may be advantageous to encourage compensatory movements in a manner that is optimally ergonomic to facilitate task execution and minimize injury, and this may have implications for prosthetic training. The recruitment of trunk and shoulder motion demonstrates the flexibility of the upper body motor system and the adaptability of transradial prosthesis users when executing ADLs. Training that encourages successful management of the DoFs of the upper body could counter the functional limitations of currently available transradial prostheses, and although compensatory movements may impose unfavorable metabolic costs, the functional improvements may ultimately enhance user perception of device utility –27. HoweApart from trunk forward flexion, the DoFs that displayed increased RoM in prosthesis users also demonstrated a significant increase in within-subject SD compared to able-bodied controls. These results suggest that these compensatory dynamics appear to be associated with an increase in average variability of movement, such that prosthesis users generally do not converge on a defined motor synergy to the degree observed in able-bodied individuals. The DoFs that did not demonstrate a significant increase in SD for prosthesis users, which would otherwise indicate greater variability, displayed a significant increase in CMD for able-bodied controls. This result suggests greater kinematic repeatability of controls compared to prosthesis users for those DoFs, and also the complementary information provided by the SD and CMD techniques. The increased shoulder and trunk variability of prosthesis users did not preclude execution of tasks 3, 4, and 5, and this further demonstrates the utility of the motor flexibility associated with the upper body kinematic chain to manipulate end point position. Consequently, this variability may be seen as advantageous when operating a transradial prosthesis with reduced active distal DoFs and reflective of healthy motor performance , 48, wheIncreased variability may also be viewed as diminished movement quality when compared to able-bodied individuals, as real-time adaptation during execution was not necessary since there were no external factors such as perturbation. Although the tasks executed in this study did not require specific joint/segment configurations, able-bodied individuals were able to maintain relatively low levels of within-subject absolute variability Figure  and whilOverall kinematic repeatability shared a direct relationship with prosthesis experience. As only prosthesis users were tested in this study and only during one testing session, the causal relationship between experience and variability is unknown, i.e., whether kinematic variability decreases over time with device experience, or low levels of variability encourage continued device use. However, motor adaptation and learning through practice and experience is essential for upper extremity neurorehabilitation to minimize variable outcomes and maximize likelihood of successful task execution . Similarin-vivo prosthetic upper limb research given a small overall population size, prosthetic designs varied between participants. Although there is limited evidence to suggest that there is no difference in functional performance between single DoF and multi-articulated prosthetic hands [A limitation of this study is that due to the small sample size, which reflects the practicalities of ic hands , the useic hands , may offTransradial prosthesis users utilize shoulder abduction and trunk movement as compensatory motions to execute goal-oriented tasks, and the majority of these motions are accompanied by increased kinematic variability when compared to able-bodied controls. The average repeatability of upper body kinematics was positively associated with prosthesis experience. As these dynamics may be necessary to compensate for the absence of active distal DoFs in the prosthetic arm, transradial prosthesis users may benefit from dedicated training that: 1) encourages optimization of these dynamics to facilitate execution of ADLs, and 2) fosters adaptable but reliable motor strategies. Importantly, results from this study further emphasize the utility of motion capture for investigating movement quality of upper limb prosthesis users, which can complement information on functional clinical outcomes when assessing overall performance. As new upper limb prosthetic designs with increased active DoFs become commercially available, more sophisticated outcome metrics that consider functional clinical outcomes and movement quality may be necessary to effectively assess the contribution of these design features to user performance.


Motor neurons are cells located in specific areas of the central nervous system, such as brain cortex (upper motor neurons), brain stem, and spinal cord (lower motor neurons), which maintain control over voluntary actions. Motor neurons are affected primarily by a wide spectrum of neurological disorders, generally indicated as motor neuron diseases (MNDs): these disorders share symptoms related to muscular atrophy and paralysis leading to death. No effective treatments are currently available. Stem cell-derived motor neurons represent a promising research tool in disease modeling, drug screening, and development of therapeutic approaches for MNDs and spinal cord injuries. Directed differentiation of human pluripotent stem cells - human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) - toward specific lineages is the first crucial step in order to extensively employ these cells in early human development investigation and potential clinical applications. Induced pluripotent stem cells (iPSCs) can be generated from patients’ own somatic cells  by reprogramming them with specific factors. They can be considered embryonic stem cell-like cells, which express stem cell markers and have the ability to give rise to all three germ layers, bypassing the ethical concerns. Thus, hiPSCs constitute an appealing alternative source of motor neurons. These motor neurons might be a great research tool, creating a model for investigating the cellular and molecular interactions underlying early human brain development and pathologies during neurodegeneration. Patient-specific iPSCs may also provide the premises for autologous cell replacement therapies without related risks of immune rejection. Here, we review the most recent reported methods by which hESCs or iPSCs can be differentiated toward functional motor neurons with an overview on the potential clinical applications. Motor neurons (MNs) are differentiated cells that control voluntary actions and are affected primarily by a wide spectrum of neurological disorders, generally indicated as motor neuron diseases (MNDs). MNDs may present with a range of symptoms deriving from muscular weakness/atrophy and leading to death . CurrentEvery year, MNDs affect approximately 2 new cases per 100,000 people, and the prevalence of these disorders is about 5 to 7 cases per 100,000 . The lifSMN1 gene , resulting in the selective degeneration of lower α-MNs. The SMN2 gene, an SMN1 homologue, compensates for the abnormal production of SMN1 protein, and its levels of expression correlate with disease severity . Mo. Mo26]. The term neuralization refers to the specification of the ectodermal cells toward epidermis or neural fate. This specification is influenced by the bone morphogen protein (BMP) that belongs to the transforming growth factor-beta (TGF-β) superfamily of proteins . TherefoBMP factors are selectively located in the ‘roof plate’ regions of the dorsal neural tube. Other growth factors are expressed specifically by the most ventral region (‘floor plate’). The roof plate gives rise to the interneurons, whereas the region of floor plate is the site of origin of developing MNs. The dorsoventral axis of the neural tube is established by Wnt morphogenetic signaling gradients: high concentration of Wnt determinates the dorsal region, whereas Shh signaling defines the ventral region . Wnt proin vitro some of these physiological steps in order to obtain enriched MN populations starting from human iPSCs and ESCs. At present, there are many protocols that exploit morphogens or synthetic agonists to promote MN specification [Understanding pathways and specific morphogens that regulate MN development is crucial to replicate fication ,38.Different methods for MN differentiation have been performed Table ; here, wThe term neural induction refers to the formation of neural progenitors deriving from pluripotent stem cells. There are several methods to promote neural induction; the majority include neural rosette and EB formation Figure .Neural rosettes can be considered a stage at which neural stem cells exhibit a high proliferative potential and broad differentiation capacity along both neuronal and glial lineages in response to appropriate developmental signals . Neural Reinhardt and colleagues  reportedIn recent years, many groups have exploited protocols focused on the formation of EBs to differentiate stem cells into MNs. EBs are three-dimensional cell aggregates that grow spontaneously, self-assembling in suspension cultures. For EB formation, iPSCs or ESCs are detached from mouse embryonic feeder fibroblasts and cultivated onto ultralow adherent culture dishes. Cell culture medium is similar to the one used for hESC culture, supplemented with TGF-β and BMP inhibitors. Media usually contain Noggin or a small-molecule substitute, like Y-27632  to increSome authors adopted a combination of the two methods: EBs were cultivated in a suspension environment and then the aggregates were dissociated and seeded on laminin/poliornithin-coated plates to form neural rosettes ,43.in vitro appears to summarize the sequence of events physiologically involved in spinal cord development. In 2002, Wichterle and colleagues [+, Hb9+, Islet1+, and choline acetyltransferase-positive (ChAT+) neurons. Transcriptional upregulation of MN markers such as Islet 1, Hb9, and Olig2 was shown. Terminally differentiated neurons were Synapsin-positive and electrophysiologically active. MNs were capable of recreating neuromuscular junctions in culture with C2C12 myotubule cells. Overall, these data indicate that hESCs can differentiate into MNs that express specific molecular markers and have functional properties similar to those of physiologically developed MNs. Through the years, many reported protocols have tested different efficacious concentrations of the crucial signaling molecules, from 1 nM to 1 μM of RA and from 50 to 500 ng/mL of Shh [Thanks to the cultural background of developmental biology, it is known that MN differentiation lleagues  were thelleagues  differenL of Shh ,39,40,45Another crucial variable to be modulated is the time of addition of the small molecules for differentiation. Hu and Zhang  reportedin vitro studies and cell therapies. The manipulation of Hox gene network specifying columnar and pool MN identity may be fundamental to selectively modulate the differentiation of precursor cells into specific MN subtypes. Recently, Amoroso and colleagues [in vivo. However, it has to be considered that this marker does not provide any information on motor neuronal subtype and the fluorescence of GFP could give false-positive results due to the GFP long half-life. Very few GFP+ cells were observed in the absence of exogenous Shh agonists. Recombinant Shh and human-specific Smo agonist each gave rise to less than 10% GFP+ cells. In contrast, the Smo agonist (SAG) alone gave rise to 16% ± 4% GFP+ cells and purmorphamine alone induced 22% ± 6%. RNA-seq analyses revealed an enrichment of spinal MN markers and the expression of cholinergic genes in cells treated with SAG and purmorphamine compared with only Shh-treated ones. This work exploited non-viral protocols of differentiation, characterized also by relatively high rapidity and efficiency. Not less important, Amoroso and colleagues [Another variable to be considered is the natural habit of human stem cells to differentiate into caudal/rostral subtypes giving rise to medial/lateral column as well as cranial MNs. Similarly, the selective differentiation signal inducing the development of specific neuronal subtypes within the wide range of spinal cord MNs warrants further studies to be extensively applied in lleagues  systematlleagues  made greMany other differentiation cocktails have been reported to share the objective of gaining highly enriched spinal cord motor neuronal populations: supplementing RA and Shh with B27 ,46, cAMPThe discovery and establishment of the use of iPSCs provided further advances in the field; it has become possible, by using hiPSCs, to produce MNs carrying the specific combination of genetic variants that caused neurodegeneration in a single patient. A multistage MN differentiation protocol starting from SMA patient-derived iPSCs has been performed by Corti and colleagues . For MN in vitro models of disease and as a feasible therapeutic approach. In regard to the former, generated MNs presented features indicative of mature lineage and spinal MN commitment such as HB9/ISLET1 and SMI32. The in vitro differentiation protocol generated a mixed cell population, including non-motor neuronal cells. To further isolate and purify MNs, a physical strategy based on gradient centrifugation and leading to an enriched MN population was exploited [This study provided important data on the potential applications of stem cells both as xploited . MoreoveSimilarly, different methods have been performed for MN induction starting from hiPSCs derived from patients affected by ALS. Sareen and colleagues  generatein vitro modeling, it is first necessary to evaluate their proper differentiation, assessing that they have the same features as MNs in vivo. An efficient MN differentiation starting from human ESC and iPSC lines can be assessed by evaluation of those features that are the hallmarks of MNs in vivo   Figure . The aute in vivo-27. Indee in vivo,39,42,45Karumbayaram and colleagues  demonstrIn several studies, stem cells were manipulated in order to express GFP driven by an MN-specific reporter. A reporter specific for activity of Hb9 which encodes for a transcription factor specifically expressed by mature MNs is most commonly used because of its relatively high specificity and assessed staining.in vivo. Hb9:GFP stem cells can be studied for their maturation in cellular morphology  and increased soma area over time [in vivo: spike frequency adaptation and rebound action potential firing [Hb9-driven GFP reporter is usually transfected into stem cells, allowing the identification of MNs in which Hb9 is transcriptionally active. This technique facilitates cell study in culture and cell tracing ver time ,39,44. Over time . Electrover time ,38,39,49ver time  observedl firing .In vivo morphological analyses can consist of xenotransplants by injection of stem cell-derived MNs  into animal models. Pluripotent stem cell-derived MNs demonstrated the ability to project axons outside of the CNS through the ventral  roots and to follow proper neural paths when transplanted into spinal cord of developing chickens [chickens  or mice chickens ,29. Furtchickens  demonstrin vitro basic disease processes. Stem cells could also be manipulated in order to express mutant genes related to MNDs [Disease modeling represents an interesting possibility of application of human stem cell-derived MNs: with the optimization of standardized protocols for stem cell differentiation, it has become achievable to produce human MNs in substantial amounts for elucidating  to MNDs ,55.The discovery of reprogramming methods using specific factors and the subsequent derivation of the first patient-specific hiPSC lines provided a further step in this direction.in vitro and thus address specific questions about altered neuronal differentiation and function that might cause the development of diseases such as ALS, SMA, and many others. The possibility to recreate in vitro a reliable model of human disease is valuable, as there have been many examples of therapeutic approaches that were relatively efficacious in animal models but unfortunately did not translate well to patients [iPSCs can be employed to derive human MNs patients .de novo TDP-43 aggregation and that these aggregates summarized pathological features in post-mortem tissue from one of the three patients from which iPSCs were derived. The authors then performed a high-content chemical screen by using the TDP-43 aggregate endpoint in both lower MNs and upper derived MNs identifying US Food and Drug Administration-approved small-molecule modulators.Burkhardt and colleagues  reprograCorti and colleagues  generateThus far, various iPSC lines from patients with various neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, have been generated -58. Pati+ fibers. These data suggest that hESC-derived MNs may be able to project toward the ventral root through the adult spinal cord, similarly to the embryonic chick spinal cord even if with a different time scale. This study provided evidence for in vivo survival of hESC-derived MNs, a crucial requirement for future preclinical applications.Cell therapy in neurodegenerative disorders acts by introducing functional cells in order to rescue the function of damaged neural tissues. Transplantation of hESC-derived MNs into the developing chick embryo resulted in correct engraftment, maintenance of motoneuron identity and long-distance axon elongation outside the CNS, reaching properly peripheral muscular targets . TransplSMN2 gene was converted into an SMN1-like gene with a strategy based on the use of single-stranded oligonucleotides [In vivo experiments after transplantation into the spinal cord of transgenic SMA mice showed that iPSC-derived MNs can survive and integrate into the spinal cord of SMA mice and ameliorate the SMA type I phenotype. Transplanted SMA mice had a longer survival (about 50%) compared with vehicle-treated mice, a beneficial effect that was more relevant with treated SMA-iPSC MNs than with untreated ones [hiPSCs are similar to hESCs, and the experimental strategies developed for hESCs could be applied to iPSCs without major modifications. In addition, hiPSCs do not have issues in the immunologic compatibility between donors and recipients reported in hESCs . Thus, tleotides . In vivoted ones .These appealing premises for clinical therapies have to be modulated by the presence of crucial issues to be addressed before effectively translating cell-mediated approaches to the clinic. Important concerns are related to the generation and preparation of cells under good manufacturing practice (GMP). It has been demonstrated that ESCs can be generated without the use of reagents of animal origin (xenobiotics); however, the derivation of patient-specific iPSCs usually requires methods of reprogramming that are not performed in xeno-free conditions. iPSCs generated with viral methods present major risks related to the stochastic activation/inactivation of endogenous genes. Non-integrative methods  have been improved to bypass this concern and facilitate the transition to the clinic . Neural Concerns related to the host immunorejection could be bypassed with the use of autologous cells, such as iPSCs. However, studies rigorously assessing iPSC immunogenicity still need to be performed, and results could vary on the basis of cell preparation protocols.in vitro studies at first and then by a rigorously assessed clinical trial in humans.The clinical perspectives on the use of cell-based therapies have to be calibrated on the basis of the previous issues: the premises for future treatments are great but need to be thoroughly tested by standardized Despite many aspects to be cleared and certainly worthy of further studies , these preliminary results open the way to a future in which human pluripotent stem cells may provide a source of healthy MNs for therapeutic transplantation for SMA and other MNDs.The potential of human pluripotent stem cells to treat patients with neurodegenerative disease is enormous. Stem cells can be introduced into clinical applications in several different ways, such as disease modeling, drug screening, and cell replacement therapy.in vitro generation of large numbers of various neural cell types and for their use in the repair of the nervous system. Advances in obtaining and understanding MNs have been made in recent years; however, some aspects still need to be investigated. Among them, the molecular signature of MN development and specification into different subtypes in vivo is only partially known. In regard to different methods exploited to produce MNs, extensive applications need a precise consideration of the experimental conditions for cell plating and culturing. Moreover, challenges such as those ensuring clinical safety should be overcome. Even though it may take quite a long time to address all of these questions, the establishment and optimization of human stem cell differentiation protocols to develop hiPSC-based clinical applications may hold the key to curing neurodegenerative disorders.The development and optimization of non-viral methods of stem cell reprogramming will allow clinicians to bypass concerns related to random viral integration. The use of autologous patient cells could also alleviate problems associated with host immunorejection. In regard to the production of differentiated cells, it is crucial to assess protocols with high efficiency allowing a large-scale production for quality-control assessment. A very recently published study reported the generation of MNs from iPSCs in 20 days . In viewSOD1: Superoxide dismutase; TDP-43: Tar-DNA-binding protein 43; TGF-β: Transforming growth factor-beta.ALS: Amyotrophic lateral sclerosis; BDNF: Brain-derived neurotrophic factor; BMP: Bone morphogen protein; ChAT: Choline acetyltransferase; CNS: Central nervous system; EB: Embryoid body; ESC: Embryonic stem cell; FALS: Familial amyotrophic lateral sclerosis; FGF: Fibroblast growth factor; GDNF: Glial cell-derived neurotrophic factor; GFP: Green fluorescent protein; GMP: Good manufacturing practice; hESC: Human embryonic stem cell; hiPSC: Human induced pluripotent stem cell; hNPC: Human neural progenitor cell; iNSC: Induced neural stem cell; iPSC: Induced pluripotent stem cell; MN: Motor neuron; MND: Motor neuron disease; PCR: Polymerase chain reaction; RA: Retinoic acid; SAG: Smo agonist; SALS: Sporadic amyotrophic lateral sclerosis; Shh: Sonic hedgehog; SMA: Spinal muscular atrophy; SMN: Survival motor neuron; The authors declare that they have no competing interests.All authors read and approved the final manuscript.


RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA ‘specificity’ design rules. Small interfering RNAs (siRNAs) are double stranded RNA molecules designed to perfectly match the sequence of a target gene and silence its expression. The function is exerted through the RNA interference (RNAi) pathway and has revolutionised biological research due to its ease-of-use and high potency. While siRNAs were initially believed to be highly specific, they have subsequently been observed to interact with other, unintended messenger RNAs. However, the mechanistic details of this process remain poorly understood, and there is a paucity of strategies and guidelines directed toward mitigating this issue. To address this potential safety liability, we performed a comprehensive analysis of sequence characteristics of siRNA duplexes and their target regions. Results from luciferase-reporter assays and global expression data confirmed previous observations that the siRNA seed region is the primary determinant for off-target gene recognition and binding. Furthermore, our analysis revealed the important contribution of siRNA non-seed region, and its corresponding target sequences, to the potency of off-target knockdown. Similar results were observed in an equivalent evaluation of the miRNA-targeting mechanism, suggesting that the correlating features arise through an evolutionary conserved mechanistic factor. Whene strand . This elnd to 7th/8th nt in the 5’ region (seed region) of siRNA recognises the unintended target gene within the 3’UTR of mRNA sequence  of various subsections within siRNA guide strand with experimental results derived from off-target effect screens. Subsequently, we have utilized DNA microarray data generated using siRNAs that satisfy the sequence rules of highly effective siRNAs , to deteTm values of seed-target duplex and off-target silencing efficiency. Longer sequence subsections that overlap a highly correlated shorter region, are more likely to show higher correlation. It is thus appropriate to concentrate on the subsections showing the highest correlation coefficient and the shortest length. Interestingly, nucleotides 2–5 and not the whole seed (nucleotides 2–8) showed the highest positive correlation at all siRNA concentrations , suggesting that positions 2–5 may be the most crucial in the interaction between the guide strand and off-target transcripts. The correlation between 3’ termini positions and knockdown efficiencies exceeded r = 0.7 in results from assays at 0.5 and 5 nM concentrations. Asymmetry in thermodynamics of 3’ and 5’ termini bases in the siRNA duplex determines which strand will be preferential selected and loaded into RISC  is the sodium ion concentration (100 mM).Thermodynamic profiles were created by evaluating every subsection within the guide strand of siRNA . Similarly, the correlation between GC contents of all possible target sequences corresponding to the various siRNA non-seed subsections, and the fold changes derived from DNA microarray experiments, was calculated for siVIM-270 and siVIM-805. The profiles were plotted as heatmaps, as a convenient method to represent and interpret two dimensional data Click here for additional data file.


Intraosseous mucoepidermoid carcinoma is a rare tumor which affects women more than men and is more common in the mandible. The radiological examination reveals a well-defined unilocular or multilocular radiolucent lesion. This tumor may resemble a glandular odontogenic cyst, particularly in incisional biopsies. The accurate diagnosis of these lesions is imperative because the subsequent treatment of each lesion would be different. The purpose of this study is to report two cases of intraosseous mucoepidermoid carcinoma and explicate the differentiating criteria of this lesion from the glandular odontogenic cyst.  Intraosseous mucoepidermoid carcinomas (MEC) of the jaws are rare, establishing 2-3% of all testified mucoepidermoid carcinomas in the literature -2. This Intraosseous MEC affects females more than males and apparently implicates the mandible more than the maxilla . It may It has been reported in all age; it is more prevalent in 4th and 5th decades of life . Wide loGlandular odontogenic cyst (GOC), a rare lesion, constitutes 0.2% of all odontogenic cysts . This tuThe purpose of this report was to depict two cases of intraosseous mucoepidermoid carcinoma and elucidate the possible ways to differentiate this lesion from a glandular odontogenic cyst. A 44-year-old woman was referred to Jahad Clinic- Ahvaz, Iran, exhibiting a painless swelling in the left posterior region of the mandible endured for 2 years. The entire molar teeth were extracted in this region. The radiological examination illustrated a large, well-defined, multilocular radiolucent lesion with scalloped borders extending from the first molar area into the ascending ramus ; which pNo palpable lymph node was noticed in the neck on extraoral examination. A provisional diagnosis of odontogenic tumors was verified and subsequently an incisional biopsy was performed. The microscopic examination revealed islands of epidermoid, mucous and intermediate cells with cystic areas in a fibrous stroma . The finA 56-year-old edentulous woman was referred to Jahad clinic, Ahvaz, Iran,  complaining of a painless expansion of right posterior mandible endured for 11 months. Radiological assessment illustrated a large, well-defined multilocular radiolucent lesion, extending from the posterior body of the mandible into the ascending ramus . On extrThe criteria that acknowledges the diagnosis of intraosseous MEC is entailed as: (a) intact cortical plates while the cortical perforation does not exclude the diagnosis, (b) radiological evidence of bone destruction, (c) exclusion of an alternative primary tumor, which its metastasis could histologically resemble the central MEC, (d) exclusion of an odontogenic tumor and (e) histopathologic confirmation , 5. In tThe radiological features are diverse and presumably non-diagnostic, usually presented as a unilocular or multilocular radiolucency. Even though it is frequently scalloped; the margins of the lesion are often well defined . To the Chan et al.  stated tThe presence of calcifications was reported in the clear-cell variant and the conventional MEC, occurring in enduring neoplasms. The dystrophic calcification of the amorphous-eosinophilic material secreted by intermediate basal cells may perhaps produce these features -15. EverHistopathologically, GOC may be confused with intraosseous MEC -9. This Pires et al.  stated tThey compared CK expression in GOC and central MEC and noticed dissimilarities in CKs 18 (30% versus 100%) and 19 (100% versus 50%). Central MEC and GOC are perhaps distinct lesions with CK profiles comparable to lesions that have glandular and odontogenic origins, respectively. Moreover, expression of CKs 18 and 19 could be beneficial in their subsequent differential diagnosis .TORC1/MAML2 and MECT1:MAML2 gene fusion in intraosseous MEC was reported by studies of Khan et al.  and FowlThe foremost treatment for intraosseous MEC is wide local resection, enblock resection or hemi-mandibulectomy , 20. SelRadiotherapy seems to be a useful supplementary aid in cases represented with close surgical margins and high grade tumors . MicroscIn He et al.’s study , all patThe recurrence rate of this entity varies from 13-50% in different studies . MetastaIntraosseous MEC may resemble GOC; therefore, pathologists and surgeons should deliberate the diagnosis of central MEC in their mind once the pathology report signifies GOC. This is particularly imperative when the lesion is located in the posterior of the mandible. Although many case reports outlines the radiological features of this entity, the diagnosis should be established on clinical and pathologic characteristics and local or radical resective surgery would probably be the first option for patient treatment. Immunohistochemistry might be useful in the diagnosis of complicated cases or small specimens. Concerning the diagnosis of GOC, obtaining serial sections of the lesion is highly recommended.


Cancer Immunology Research, antibody biomarkers were screened in pancreatic cancer patients who received allogeneic, granulocyte-macrophage colony stimulating factor-secreting pancreatic cancer vaccine (GVAX) by using a serum antibody-based SILAC immunoprecipitation (SASI) approach. Using this assay, several new tumor antigens  were identified that were found to have significantly different expression in tumors compared with normal tissue. Moreover, patients with detectable antibodies showed improved disease-free survival after GVAX therapy. These targets need to be further validated to determine the full spectrum of tumor antigen immunogencity and their potential clinical application. In addition to antibodies, circulating protein, DNA and RNA in peripheral blood are under clinical investigation as liquid biopsies and have the potential to provide guidance for future personalized cancer immunotherapy.Immune checkpoint blockade therapies are revolutionizing standard cancer treatments. Immune checkpoint inhibitors likely function to enhance the tumor specific antigen response in order to achieve favorable clinical outcomes. Thus, continuous efforts to identify the common tumor-specific antigens are essential for the broad clinical application of these therapies. Several immunoproteomics approaches have been used in order to screen for this specificity. In a recent article from Jhaveri and colleagues published in the February issue of  Immune checkpoint blockade therapies are revolutionizing standard cancer treatments . It is aSerologic Proteome Analysis (SERPA), Serological analysis of recombinant cDNA expression libraries (SEREX) and protein microarrays have been explored extensively to find tumor associated antigens and their cognate antibodies [It is a long standing interest in the potential of antibodies to search for tumor specificity. As Dr. Lloyd J Old highlighted in his G.H.A Clowes Memorial lecture three decades ago, “Antibodies might reveal something specific, something unique about the cancer cell…The antigenic determinants were recognized by antibody” . Severaltibodies . SERPA iProtein microarrays are based upon the development of DNA microarray techniques and have thousands of purified proteins immobilized on a solid surface. Protein microarrays are generally classified into three types: analytical, functional and reverse-phase protein microarrays. ProtoArray®, a functional array, offers a unique approach to analyze the serological response against thousands of protein at the same time. This approach is limited by the number of proteins coated on the slides  .Cancer Immunology Research [In the February issue of Research , JhaveriIn this study, pre-vaccine sera was intentionally subtracted from post-vaccine sera in order to assess the vaccine-induced specific antibody responses. In doing so, a few antibodies were identified as targets from post-vaccination samples in patients with favorable clinical outcome. The expression of three antigens  was measured in tumor and normal duct epithelium, and significant differences were found in the expression of these three antigens in tumor compared with normal tissue. Moreover, patients with detectable identified antibodies showed improved disease-free survival.Overall, the SASI approach was found to identify new tumor antigens as potential biomarkers and therapeutic targets. This approach could also be applied to other similar clinical studies without protein synthesis, but these new targets require further validation as possible pancreatic cancer biomarkers. The caveat and potential limitation of this study is the subtraction of pre-vaccine sera. It limits the ability to identify the baseline antibody response, which may predict the patient’s response to GVAX vaccination. In addition, allogeneic tumor cells instead of autologous tumor cell lines were used for the vaccination. Thus, targets from autologous tumor cells may be partially missed because of the limitation of the allogeneic tumor immunogenicity profile.+ T-cell responses is frequently observed with other tumor antigens. CTLA-4 blockade induced a broad antibody response in cancer patients with ovarian, prostate cancer and melanoma [+ T cells did not experience clinical benefit. Therefore, cellular tumor antigen-specific CD4+ and CD8+ T cell response needs to be evaluated to obtain the full spectrum of the identified antigens immunogencity and explore the potential clinical application.The SASI approach is an effective method to identify tumor-specific antigens, especially common tumor rejection antigens that would allow for the development of “off-the-shelf” vaccinations. In addition to the validation of the expression and distribution of these new targets, it is of importance to further characterize these antibodies and the antigen-specific CD4+ and CD8+ T cell response. The dissociation between antibody responses and antigen-specific CD8melanoma , 10. Advmelanoma . The majAntibodies are useful for the discovery of tumor-specific antigens. Moreover, antibodies may be able to directly or indirectly eliminate tumor cells through opsonization, antigen presentation to T cells and by initiating NK cells or complement-dependent cell toxicity . SeveralIn addition to antibodies, proteins circulating in blood could be potential biomarkers for cancer immunotherapy. As an example, patients with low baseline vascular endothelial growth factor (VEGF) experienced better clinical outcome in advanced melanoma patients treated with ipilimumab. Thus, serum VEGF may be a predictive biomarker for ipilimumab treatment . With adBesides proteins and antibodies, tumor cells can also release DNA and RNA into the blood by a variety of microvesicle-dependent or -independent mechanisms. Novel technologies with minimal specimen requirements allow the characterization of these tumor derived DNA and RNA. Liquid biopsies including cell-free circulating tumor DNA (ctDNA) from plasma have been investigated for non-invasive detection and monitoring of patient tumors as well as potential biomarkers for cancer immunotherapies , 17. The


Due to indirect modulation of dopamine transmission, adenosine receptor antagonists may be useful in either treating cocaine use or improving disrupted cognitive-behavioral functions associated with chronic cocaine use. To compare and contrast the stimulant effects of adenosine antagonism to direct dopamine stimulation, we administered 150 mg and 300 mg caffeine, 20 mg amphetamine, and placebo to cocaine-dependent vs. healthy control subjects, matched on moderate caffeine use. Data were obtained on measures of cardiovascular effects, subjective drug effects , and a probabilistic reward-learning task sensitive to dopamine modulation. Levels of salivary caffeine and the primary caffeine metabolite paraxanthine were obtained on placebo and caffeine dosing days. Cardiovascular results revealed main effects of dose for diastolic blood pressure and heart rate; follow up tests showed that controls were most sensitive to 300 mg caffeine and 20 mg amphetamine; cocaine-dependent subjects were sensitive only to 300 mg caffeine. Subjective effects results revealed dose × time and dose × group interactions on the ARCI A, ARCI LSD, and VAS ‘elated’ scales; follow up tests did not show systematic differences between groups with regard to caffeine or d-amphetamine. Large between-group differences in salivary paraxanthine  levels were obtained under both caffeine doses. The cocaine-dependent group expressed significantly higher paraxanthine levels than controls under 150 mg and 3–4 fold greater levels under 300 mg at 90 min and 150 min post caffeine dose. However, these differences also covaried with cigarette smoking status , and nicotine smoking is known to alter caffeine/paraxanthine metabolism via cytochrome P450 enzymes. These preliminary data raise the possibility that adenosine antagonists may affect cocaine-dependent and non-dependent subjects differently. In conjunction with previous preclinical and human studies, the data suggest that adenosine modulating drugs may have value in the treatment of stimulant use disorders. Chronic cocaine use results in measurable disruptions in the (DA) dopamine system. Allostatic shifts in neural and behavioral systems result in neurobehavioral states that foster continued use and relapse . Accordi1 and A2 receptor antagonist that appears to modulate dopamine function at both presynaptic and postsynaptic sites   found laIn stimulant abusing subjects, both methylphenidate and d-amphetamine produced dose-dependent stimulant-like effects  . We did Previous work using the probabilistic feedback task reveals that the task is sensitive to manipulations via dopaminergic drugs ,32. For Perhaps the most intriguing and consistent trends in the data were the differences in expression of paraxanthine and caffeine levels between the groups. While no differences in caffeine levels were observed between the groups, the cocaine-dependent group showed much higher levels of salivary paraxanthine. These differences in paraxanthine levels were dose related in both magnitude and time course , and mayOne major limitation in this study was that nicotine smoking was confounded with group status (In addition to control for nicotine use rates between the groups, several other limitations must be considered. All subjects were moderate caffeine users to facilitate matching of groups, but inclusion of a broader range of caffeine use, from those who are minimal users to those who are caffeine dependent would arguably render more informative data with regards to differences between cocaine-dependent and control subjects, and variation in the effects of adenosine in cocaine dependence. The relatively small sample size likely contributed to variability in outcomes across measures. Our power to detect significant effects was clearly limited with sample sizes of 13 and 10. Another limitation of the sample size was the inability to examine gender differences, as gender and menstrual cycle phase are known to modulate the acute effects of d-amphetamine ,66. HaviWe anticipated differential change between the groups when comparing d-amphetamine and caffeine, due to changes in dopamine function that accompany chronic cocaine use and possible cross-tolerance between cocaine and d-amphetamine . However


Inhibitory neural networks have the capacity to fire synchronously depending strongly on synaptic current dynamics. Various types of inhibitory interneurons, including some with adaptation currents, are present in hippocampal circuits and are implicated in governing overall network pattern formation. However, the contribution of intrinsic cell firing patterns of interneurons to these patterns has not been fully investigated. Understanding how both synaptic and cellular properties contribute to the propensity for inhibitory neural networks to synchronize is thus an invaluable tool for investigating hippocampal network pattern formation. Through numerical simulation of large, spiking neuron, inhibitory networks, we investigate the role of the slow, adapting M-type K+ current in network pattern formation. This adapting current plays a role in hippocampal interneurons, including the OLM cells, in which the blockade of M-current by acetylcholine or other neurotransmitters switches the neuronal firing rate-current relation (f-I curve) from Type II to Type I. Other types of interneurons, such as fast-spiking PV cells, display Type II f-I curves without any adaptation current. Thus, we consider networks of three cell types: Type I neurons and Type II neurons that either contain  or do noTo probe the interaction of cellular and synaptic properties influencing synchronization in these networks, we vary the time constant of decay of the inhibitory synaptic current and the intrinsic cellular frequencies by varying the mean of the distribution of external currents applied to each neuron. With sparse, nearest neighbor connection topologies, Type I networks exhibit stationary activity patterns reminiscent of standing waves while Type II neurons exhibit traveling wave activity patterns that sweep across the network. These traveling waves are much more robust and regular when the Type II neurons contain the adaptation current. With sparse random connectivity, Type II networks without an adaptation current exhibit cluster-firing patterns, in which cells segregate into multiple clusters that show synchrony within the cluster but not across clusters. In contrast, Type II networks with the adaptation current display full synchronization for some parameter ranges, but do not exhibit robust clustering Figure . Prelimi