The lithium ion conductivity and salt diffusion coefficient of PEO18LiTFSI were dramatically enhanced by the addition of TEGDME. The water-stable lithium electrode with PEO18LiTFSI-2TEGDME, as the protective layer, exhibited a low and stable electrode resistance of 85 Ω·cm2 at 60 °C, after 28 days, and low overpotentials of 0.3 V for lithium plating and 0.4 V for lithium stripping at 4.0 mA·cm−2 and 60 °C. A Li/PEO18LiTFSI-2TEGDME/LTAP/saturated LiCl aqueous solution/Pt, air cell showed excellent cyclability up to 100 cycles at 2.0 mAh·cm−2.The electrochemical properties of a composite solid polymer electrolyte, consisting of poly(ethylene oxide) (PEO)-lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) and tetraethylene glycol dimethyl ether (TEGDME) was examined as a protective layer between lithium metal and a water-stable lithium ion-conducting glass ceramic of Li The aqueous lithium-air battery system, proposed by Imanishi and co-workers, is composed of a lithium metal electrode, a lithium ion conducting polymer electrolyte based on poly(ethylene oxide) (PEO)x-lithium bis(trifluoromethanesulfonyl)imide (LiTFSI), a water-stable lithium ion-conducting glass ceramics of Li1x+y+1x−AlxPy3−SiyO12 (LTAP), an aqueous electrolyte with saturated LiCl, and a carbon air electrode [−4 S·cm−1 at ambient temperature, and is stable in saturated LiOH and LiCl aqueous solution, but exhibits poor chemical stability towards lithium metal [Aqueous lithium-air batteries have been considered as a promising electrochemical energy storage system for electric vehicles. The theoretical energy density for the cell reaction of 4Li + 6Hum metal . The two18LiTFSI reduced the interface resistance and suppressed lithium dendrite formation [et al. [et al. reported that the addition of poly(ethylene glycol) dimethyl ether (PEGDME) into propylene carbonate (PC) with LiClO4 suppressed dendrite formation [et al. reported that the oxidative stability of glyme molecules is enhanced by complex formation with LiTFSI in a molar ratio of 1:1 [et al. [3SO3/carbon, O2 cell capable of operating over many cycles with capacity and rate values as high as 5000 mAh·g−1 and 0.5 A·g−1, respectively. We have previously reported an increase in the lithium ion transport number and decreases in the interface resistance of the lithium metal and polymer electrolyte by the addition of PEGDME to PEO18LiTFSI, where the mean molecular weight of PEGDME was 500 [et al. [4 decreased with a decrease in the molecular weight of PEGDME in the range of 90–400. In this study, the interface resistance between lithium and the polymer electrolyte, and the transport properties of the PEO18LiTFSI-xTEGDME (M = 222.28 g·mol−1) composite polymer electrolyte (CPE) are examined as a function of the amount of TEGDME (x). In addition, the electrochemical performance of a Li/PEO18LiTFSI-2TEGDME/LTAP/saturated LiCl aqueous solution/Pt, air cell is evaluated at 60 °C. In our previous studies ,7,8, we ormation . The add [et al.  reportedormation . Moreoveo of 1:1 . Recentl [et al.  demonstr was 500 . Naoi et [et al.  conclude18LiTFSI-xG4 (TEGDME abbreviated as G4 hereafter) were measured as a function of x (the molar ratio of G4 to ethylene oxide). 18LiTFSI-xG4. The conductivity increases significantly with increasing x up to 2 and there is little difference in conductivity between the samples with x = 2 and 3. The electrical conductivity of PEO18LiTFSI-2G4 at 25 °C is almost one order of magnitude higher and that at 60 °C is three times higher than that of PEO18LiTFSI. TEGDME loosens the coordination of lithium ions with EO units in the PEO matrix and thus enhances the mobility of ions, and could also enable lithium ions to decouple from ion pairs, as shown by Kriz et al. [18LiTFSI-xG4 at high temperature decrease with increasing x. That of PEO18LiTFSI-2G4 was calculated to be 25.3 kJ·mol−1, in the range of 55 to 80 °C, which is lower than that for PEO18LiTFSI (38.8 kJ·mol−1) and that for PEO18LiTFSI-2.0 N-methyl-N-propylpiperdinium bis(fluorosulfonyl) imide (PP13FSI) (33.8 kJ·mol−1) [The electrical conductivities of PEOz et al. . The actJ·mol−1) .18LiTFSI/LTAP/Pt, air cell at high current density, it is necessary to enhance the limiting current density of lithium-ion conducting PEO-based polymer electrolytes (Il), which is proportional to the salt diffusion coefficient and lithium ion transference number, as shown by Equation (1):Co is the initial concentration, e is the elemental charge, D is the salt diffusion coefficient, ta is the anion transport number, and L is the thickness of the electrolyte. The salt diffusion coefficient of PEO18LiTFSI-xG4 was estimated using the method proposed by Ma et al. [x. Typical curves for the natural logarithm of potential versus time for the Li/PEO18LiTFSI-xG4/Li cells at 60 °C are shown in D) of PEO18LiTFSI-xG4 was calculated from the slope of the linear curves using Equation (2):D are summarized in −7 cm2·s−1 was determined for x = 2.0, which is almost one order of magnitude higher than that for PEO18LiTFSI (3.60 × 10−8 cm2·s−1) and PEO18LiTFSI-1.44PP13FSI (2.25 × 10−8 cm2 s−1) [18LiTFSI-18 wt % PEGDME (8.38 × 10−8 cm−1 s−1) [The overpotentials between lithium metal and polymer electrolytes for lithium deposition and stripping generally increase with current density and undergo a sudden increase with the polarization period at a current density (limiting current density). When the ionic concentration in the vicinity of cathode drops to zero, the limiting current density is reached and dendrite growth is initiated (Sand time) . To opercm2 s−1) , and foum−1 s−1) . xG4 were measured using the method reported by Evans and Vincent [ta was calculated using the Evans and Vincent equation, and the results are summarized in i.e., decrease of the anion transport number) was observed by addition of G4 into PEO18LiTFSI. The lithium ion transport number for PEO18LiTFSI-2G4 is slightly higher than that for LiTFSI-2G4. Based on these results for D and ta, the limiting current density (Il) for PEO18LiTFSI-xG4 was calculated and the results are summarized in L) was 100 μm. PEO18LiTFSI-2G4 exhibits the highest Il of 15.8 mA·cm−2, which is higher than that for PEO18LiTFSI by a factor of approximately 15. The high limiting current density of PEO18LiTFSI-2G4 suggests that a WSLE with PEO18LiTFSI-2G4 could be operated at a high current density.Lithium ion transference numbers for PEO18LiTFSI- Vincent . Figure 18LiTFSI-xG4/Li cells at 60 °C as a function of storage time. PEO18LiTFSI-G4 and PEO18LiTFSI-1.5G4 show a significant increase of interface resistance with storage time; the initial cell resistance of 69 Ω·cm2 increased to 141 Ω·cm2 for PEO18LiTFSI-G4 after 28 days. However, low interface resistance was obtained for PEO18LiTFSI-2G4 and PEO18LiTFSI-3G4. After 28 days, PEO18LiTFSI-2G4 and PEO18LiTFSI-3G4 showed interface resistances of 34 and 74 Ω·cm2, respectively, which are much lower than 253 Ω·cm2 for PEO18LiTFSI after storage for 28 days [18LiTFSI-2G4 was unusual, where the interfacial resistance increased during the initial seven days and then decreased continuously to a value lower than the original. A similar change in the interface resistance was reported for the Li/PEGDME-LiTFSI/Li cell by Bernhard et al. [18LiTFSI-xG4/Li cells as a function of x. The activation energy for the inverse of the passivation film resistance (Rp) decreased from 76.2 kJ·mol−1 for PEO18LiTFSI to 54.7 kJ·mol−1 for PEO18LiTFSI-2G4. A decrease was also observed for PEO18LiTFSI-G4 and PEO18LiTFSI-3G4, but it was not as significant as that for PEO18LiTFSI-2G4. The activation energies for the inverse of the charge transfer resistance (Rc) decreased with increasing x. The lowest value of 63.4 kJ·mol−1 was calculated for PEO18LiTFSI-3G4, in comparison with 81.7 kJ·mol−1 for PEO18LiTFSI and 68.1 kJ·mol−1 for PEO18LiTFSI-2G4. These results suggest that the low-molecular weight oligomer ether G4 could reduce the resistance of the SEI and facilitate the charge transfer reaction at the interface. The former role is similar to other additives, such as nanofillers and ionic liquids, whereas the latter role is only performed by G4. Low and stable interface resistance between lithium metal and polymer electrolytes is essential for the electrochemical performance of WSLEs and to reduce lithium dendrite formation . Figure  28 days . The intd et al. , which cd et al. . These i18LiTFSI-2G4 motivated us to examine its role as the protective layer in a WSLE. The impedance of the Li/PEO18LiTFSI-2G4/LTAP/sat. LiCl aqueous solution/Pt, air cell was measured at 60 °C, using a platinized platinum reference electrode. 2 was obtained, which is comparable with those reported previously; the cell resistance for the Li/PEO18LiTFSI/LTAP/1 M LiCl/Pt, air cell was 539 Ω·cm2 [2 for the Li/PEO18LiTFSI-40 nm BaTiO3/LTAP/1 M LiCl aqueous solution/Pt, air cell [2 for the Li/PEO18LiTFSI-1.44 N-methyl-N-propylpiperdinium-bis(trifluromethanesulfonyl)imide/LTAP/1 M LiCl aqueous solution/Pt, air cell [Rb), the interfacial resistance between the polymer electrolyte and lithium metal electrode (Rf1), the interfacial resistance between the polymer electrolyte and the LTAP plate (Rf2), the charge-transfer resistance (Rc), and the Warburg impedance (W1) [Rb was quite stable at around 38 Ω·cm2 during the storage period. The fitting results suggest that Rf2 increased from 19.4 to 29.3 Ω·cm2, Rc decreased from 18.0 to 7.4 Ω·cm2, and Rf1 increased from 3.8 to 5.8 Ω·cm2 over the 28-day storage period.The high lithium ionic diffusion coefficient and low interface resistance with lithium metal for PEOair cell  and 130 nce (W1) . Rb was 18LiTFSI-2G4 and LTAP was investigated in an aqueous electrolyte using platinized platinum electrodes as the counter and reference electrodes. 18LiTFSI-2G4/LTAP/1 M LiCl-4 mM LiOH aqueous solution/Pt, air cell at current densities in the range of 0.5 to 4.0 mA·cm−2 at 60 °C. This WSLE exhibited quite low lithium plating and stripping overpotentials at high current densities up to 4.0 mA·cm−2. The lithium stripping and plating overpotentials at 1.5 mA·cm−2 were 0.10 and 0.15 V, respectively, which are lower than the best results obtained for the Li/PEO18LiTFSI-18 wt % PEGDME/LTAP/1 M LiCl-4 mM LiOH aqueous solution/Pt, air cell (0.29 V for plating and 0.21 V for stripping) in our previous studies [18LiTFSI-2G4.The electrochemical performance of a WSLE protected by PEO studies . The low18LiTFSI-2G4, a long period of lithium deposition at a constant current of 1 mA·cm−2 was performed using the Li/PEO18LiTFSI-2G4/LTAP/saturated LiCl aqueous solution/Pt, air cell, where the thickness of PEO18LiTFSI-2G4 was around 100 μm and the impedance of the WSLE was measured at every 4 h polarization. versus time curves as a function of the polarization period. The lithium electrode potential increased suddenly after a 25 h polarization. This potential increase may be due to lithium deposition on LTAP by lithium dendrite formation, which would result in the formation of a high resistance layer by the reaction of lithium and LTAP [2 by the short circuit of the lithium metal electrode with LTAP. The short circuit period of 25 h is approximately 2.6 times longer than that measured for a Li/PEO18LiTFSI/LTAP/10 M LiCl-4 mM LiOH aqueous solution/Pt, air cell [18LiTFSI-2G4/LTAP/saturated LiCl aqueous solution/Pt, air cell could be explained by the high lithium ion transport number, high salt diffusion coefficient, and low interface resistance [To study the lithium dendrite formation at the interface of Li and PEOair cell . The lonsistance ,23.18LiTFSI-2G4/LTAP/saturated LiCl aqueous solution/Pt, air cell for lithium deposition and stripping at a constant current density of 1.0 mA·cm−2 was measured at 60 °C, where the current was passed for 2 h. The cell voltage versus time profile is shown in 18LiTFSI-2G4. The cyclability of the Li/PEOv ≈ 600,000 g·mol−1) and LiTFSI  were dried overnight under vacuum at 50 and 160 °C, respectively, and then transferred to an Ar-filled glove box without exposure to air. G4  was immersed into activated 0.3 nm molecular sieves for 1 week and the same pretreatment by molecular sieves was performed 3 times before G4 was stored in a glove box. The composite polymer electrolytes of PEO18LiTFSI-xTEGDME in the range of x = 0–3 were prepared using a casting method [ca. 100 μm thick.PEO  in the frequency range from 0.1 Hz to 1 MHz at temperatures in the range of 25–80 °C. Z-plot software was employed for data analysis.ca. 250 μm thick) was supplied by Ohara Inc., Japan. The electrical conductivity of the LTAP plate at 60 °C was 1.4 × 10−3 S·cm−1. A Li/CPE/Li cell was assembled to investigate the interfacial resistance between lithium and the CPE. The active surface area was 1.00 cm2. The WSLE was prepared by sandwiching lithium metal foil (200 μm thick) with a Cu thin film lead, CPE, and the LTAP plate in a plastic envelope. The envelope was evacuated, heat-sealed apart from a 0.35 × 0.35 cm2 area, and then subjected to an isostatic pressure of 150 MPa for 15 min to ensure good contact. The WSLE was immersed into an aqueous solution of 1 M (or 10 M) LiCl with 4 mM LiOH. A platinized Pt electrode was used as both the counter and reference electrodes. Electrochemical measurements were conducted using a multichannel potentiostat-galvanostat (Biologic Science Instruments VMP3). The lithium ion transport number tLi+, was determined using the Li/CPE/Li cell with a combination of AC impedance spectroscopy and the DC polarization method, as originally proposed by Evans and Vincent, and later refined by Abraham and Jiang [et al. [The LTAP plate (nd Jiang ,25. The  [et al. . The Li/18LiTFSI-2G4 electrolyte exhibited a high lithium ion conductivity of 8.91 × 10−4 S·cm−1, a high diffusion coefficient of 3.37 × 10−7 cm2·S−1 at 60 °C, and a low interfacial resistance in contact with lithium metal. The Li/PEO18LiTFSI-2G4/LTAP/saturated LiCl aqueous solution/Pt, air cell exhibited excellent cyclability. The overpotentials for lithium deposition and stripping at 2.0 mAh·cm−2 for 2 h polarization at 60 °C were increased slightly from 0.10 to 0.15 V and from 0.08 to 0.13 V after 100 cycles, respectively. Therefore, the CPE is an attractive material for the interlayer between lithium metal and LTAP for the water stable lithium electrode in lithium-air batteries.The PEO


Theoretical observations based on density functional theory are presented here, which model carbon-based molecular junctions as single molecules and oligomers between fragments of graphene. Electronic coupling between the molecules and the contacts is demonstrated by the formation of hybrid orbitals in the model structure, which have significant electron density on both the graphene and the molecule. The energies of such hybrid orbitals correlate with tunneling barriers determined experimentally, and electronic coupling between the two graphene fragments in the model correlates with experimentally observed attenuation of transport with molecular layer thickness. Electronic coupling is affected significantly by the dihedral angle between the planes of the graphene and the molecular π-systems, but is absent only when the two planes are orthogonal. Coupling also results in partial charge transfer between the graphene contacts and the molecular layer, which results in a shift in electrostatic potential which affects the observed tunneling barrier. Although the degree of partial charge transfer is difficult to calculate accurately, it does provide a basis for the “vacuum level shift” observed in many experiments, including transport and ultraviolet photoelectron spectroscopy of molecular layers on conductors.Molecular junctions consisting of 2–20 nm thick layers of organic oligomers oriented between a conducting carbon substrate and a carbon/gold top contact have proven to be reproducible and reliable, and will soon enter commercial production in audio processing circuits. The covalent, conjugated bond between one or both sp The field of Molecular Electronics investigates the behavior of molecules as elements in electronic circuits, with the intent of exploiting variations of molecular structure to realize unusual electronic functions –4. Charg2 carbon substrate and aromatic molecular layer introduce a special property into carbon-based MJs, in that they contain a covalent, conjugated “contact” between two aromatic π-systems. The reproducibility, reliability, and operating life of carbon-based MJs resulted in an application in audio processing of electronic music, available commercially in 2015 as an accessory for electric guitars. The fundamental structural difference between metal/molecule and carbon/molecule MJs is expected to result in possibly significant differences in electronic behavior. In particular, the electronic interactions between the molecules and the contacts might be quite different, leading to changes in transport barriers and junction conductance ,2 –46, Si [2 , and e-b2 ,43. The Note that the HOMO and H−1 orbitals of G9–AB–G9 have energies close to that of free G9, and that the electron density is localized on the graphene fragments. The H−2 orbital of G9–AB–G9 is analogous to H−1 for G9–AB, and has electron density that extends across the AB molecule and onto both G9 fragments. 2t”, or “Hab”. For example, the “tight binding” model for off-resonance transport in molecular tunnel junctions predicts that the transport is proportional to t2(N − 1), where N is the number of “sites” between the contacts [Hab is related to charge propagation between polarons both along the chain and between different chains [t” is given by A primary motive for considering the G9–AB and G9–AB–G9 model structures is the ability to predict electronic coupling between the contacts and the molecules. Electronic coupling is a factor in most treatments of electron transfer, including Marcus theory, tunneling between two sites, and transport through numerous sites in the “tight binding” models of conducting solids. In all three cases, stronger electronic coupling increases the electron transfer and/or the conductance relevant to the chemical system considered. These theories will not be reviewed here, but all contain a factor for electronic coupling, often denoted “contacts . For trat chains –50. Thest were calculated for two G9 clusters in a vacuum, oriented in parallel or edge to edge, with the latter representing an idealized vacuum gap consisting of a G9–molecule–G9 system with the molecule absent. Several orientations are listed in tH/H−1 determined from tH/H−1 for orthogonal edge-oriented G9s is an order of magnitude lower than for parallel edge oriented planes, and both of these are approximately two orders of magnitude lower than that observed for basal-basal orientation.Values of t2 for the frontier Au orbitals in the contacts, as modulated by the intervening molecular “bridge” [t2 approximately tracked the observed conductance. We use here a conceptually similar approach for carbon-based molecular junctions by considering the electronic coupling between the G9 “contacts” of the G9–molecule–G9 system depicted in Venkataraman, et al. considered the relevance of electronic coupling in Au/molecule/Au single molecule junctions by calculating 4“bridge” . Changint values for the indicated pairs of orbitals. With the AB molecule absent, tH/H−1 for two G9s with the same spacing in edge orientation from tH/H−1 is 3 meV. Recall that the H and H−1 orbitals of G9–AB–G9 are derived from the original HOMO orbitals of G9, and the low tH/H−1 indicates weak interactions between the two G9 “contacts”. However, as is apparent in tH-2/H-3 is 111 meV, implying significantly stronger coupling compared to the same parameter for unbonded G9s (tH−2/H−3 < 0.1 meV). Note also that H−2 and H−3 in the G9–AB–G9 system have electron density on both G9 contacts as well as the molecule, which may indicate their likely involvement in transport. Also shown in tH/H−1 remains small (below 5 meV) for the full range of planar to orthogonal planes, while tH−2/H−3 decreases from 187 meV for the planar case to less than 1 meV for dihedral angles of 90°. In our previous theoretical analysis of electronic interactions between the larger G54 graphene fragment and AB, we proposed that the tunneling barrier is related to the offset between the G9–AB HOMO and the G9–AB orbital having significant electron density on the AB molecule [Consider first the G9–AB–G9 model of molecule . This pomolecule ,51–52. Fn–G9 were determined for n = 1–5. Selected orbital energies and t values appear in tH−2/H−3 between the optimized and planar geometries are small compared to the difference between the presence and absence of the AB molecule.The model was extended to oligomers of AB between the G9 contacts, in order to assess the effect of molecular layer thickness. Oligomers were assumed to be bonded at the ends of the AB molecules  and the optimized structures of G9–(AB)In order to assess the effects of molecular structure on electronic coupling, several series of oligomers between G9 fragments were calculated, which differ in the degree of conjugation, including alkanes, alkene, and alkynes, with the results summarized in tH−2/H−3 for the molecules listed in tH−2/H−3 covers a wide range from below 1 meV to above 700 meV. With the exceptions of alkenes and alkynes, tH−2/H−3 decreases as the separation of the G9 “contacts” increases, and logically must reach zero at infinite molecular layer thickness. The increase in coupling with distance calculated for alkenes and alkynes is unexpected, although it is small compared to the differences between alkanes, vacuum, and aromatics. The alkene and alkyne cases are not readily accessible with carbon contacts, hence this unexpected result is difficult to test experimentally. As suggested by a reviewer, it may be an indication that the electronic coupling represented by tH−2/H−3 is not due simply to coupling between the G9 contacts (which should certainly decrease with distance), but might instead indicate coupling between molecule and G9 orbitals. This possibility is testable by further experiments with variations in molecular structure. For the readily fabricated aromatic molecular junctions, it is likely that the particular orbitals most involved in transport will be revealed by further correlations of calculated orbital energy splitting with experimental results.As was the case with AB, similar trends were observed for both the planar and optimized structures, as shown in Table S2 . Figure −1) than conjugated systems (β = 2–4 nm−1), and many of the latter have similar β values despite structural differences. AQ is an interesting case, because is it “cross conjugated” with a much smaller tH−2/H−3 than that for the structurally related anthracene case [tH−2/H−3 and the attenuation coefficient β depends on the transport mechanism, the similarity between tH−2/H−3 determined for G9–molecule–G9 structures is at least a guide toward predicting transport in proposed junction structures, or for systems which are difficult to realize experimentally.The conjugated systems show similar electronic coupling, with a weaker decrease with thickness than the alkanes. The trends in electronic coupling evident in ene case . Severalene case –58. Sinc3EHOMO – EH−2 represents the magnitude of the tunneling barrier, in which case prediction of the barrier is straightforward for the model system. However, several authors have pointed out that electronic coupling between the contacts and molecules at organic/conductor interfaces can significantly perturb the simple picture, due to local electrostatic effects [− are transferred to the AB molecules, as shown in As noted in the introduction, we have examined the postulate that the energy barrier for tunneling across carbon-based molecular junctions correlates with the difference in energy between the system HOMO and the orbital closest in energy that has significant electron density on the molecule . The arg effects 40,59–60. effects ,38,40. FQ, the dielectric constant, ε, and an estimated interface thickness, δ: Δ = δQ/ε [Ef − EHOMO would decrease from the value predicted for the free molecule by 0.35 V. The assumptions required for this calculation do not permit a rigorous quantitative analysis of the effect, but it does indicate that transport barriers can be significantly perturbed by partial charge transfer. We showed previously that the effect is sufficiently large in carbon MJs with aromatic molecular layers that differences in free molecule HOMO levels caused a small effect on junction conductance. Since low HOMO levels cause larger charge transfer than higher HOMO levels, the effect “levels” a range of barriers of 0.7 to 3.0 eV based on the free molecule HOMOs to 1.3 ± 0.2 eV determined experimentally [The local electrostatic potential associated with these electrons shifts all of the orbitals in AB to higher energy, and decreases the transport barrier predicted from the work function and HOMO energy of the separated contact and molecule. Although there is some concern about the accuracy of DFT for predicting Mulliken charges , trends Δ = δQ/ε . Using tmentally .t value should promote electron transfer for both tunneling and activated “hopping” mechanisms, and decreases with increasing distance between the coupled sites. All molecules examined increase electronic coupling between graphene “contacts” compared to a vacuum gap, but the increase is strongly structure-dependent, as shown in The author agrees with the often-quoted words attributed to I. M. Koltoff: “theory guides, experiment decides”. The purpose of the current paper is to identify some of the “guides” that apply to how molecular structure affects electron transport in carbon-based molecular electronic devices. With that objective in mind, several observations are available from the DFT results. First, electronic coupling is an important factor in understanding transport, both coupling between the molecule and the contacts, and between subunits within the molecular layer. Strong coupling indicated by a large Supporting Information features the orientations of G9 fragments used to calculate the energies of File 1Additional computational data.


Bovine tuberculosis (bTB) infection is generally correlated with individual cattle’s age, sex, body condition, and with husbandry practices such as herd composition, cattle movement, herd size, production system and proximity to wildlife—including bTB maintenance hosts. We tested the correlation between those factors and the prevalence of bTB, which is endemic in Ethiopia’s highland cattle, in the Afar Region and Awash National Park between November 2013 and April 2015. A total of 2550 cattle from 102 herds were tested for bTB presence using the comparative intradermal tuberculin test (CITT). Data on herd structure, herd movement, management and production system, livestock transfer, and contact with wildlife were collected using semi-structured interviews with cattle herders and herd owners. The individual overall prevalence of cattle bTB was 5.5%, with a herd prevalence of 46%. Generalized Linear Mixed Models with a random herd-effect were used to analyse risk factors of cattle reactors within each herd. The older the age of the cattle and the lower the body condition the higher the chance of a positive bTB test result, but sex, lactation status and reproductive status were not correlated with bTB status. At herd level, General Linear Models showed that pastoral production systems with transhumant herds had a higher bTB prevalence than sedentary herds. A model averaging analysis identified herd size, contact with wildlife, and the interaction of herd size and contact with wildlife as significant risk factors for bTB prevalence in cattle. A subsequent Structural Equation Model showed that the probability of contact with wildlife was influenced by herd size, through herd movement. Larger herds moved more and grazed in larger areas, hence the probability of grazing in an area with wildlife and contact with either infected cattle or infected wildlife hosts increased, enhancing the chances for bTB infection. Therefore, future bTB control strategies in cattle in pastoral areas should consider herd size and movement as important risk factors. Mycobacterium bovis is a zoonotic disease, and remains a cause of concern for livestock, wildlife and human health [Mycobacterium bovis worldwide [M. bovis from contaminated pasture or water is also a risk factor for transmission of the disease [Bovine tuberculosis (bTB) caused by n health ,2,3. In n health . Cattle orldwide ,6,7, whiorldwide ,9,10. In disease .bTB outbreaks can trigger large economic costs to society since it can affect international trade of animals and animal products, create productivity losses , call for expensive animal market restriction measures, trigger large control and eradication programs, and increase human health costs ,12. StudStrikingly, risk factors for bTB transmission are not well known in developing countries, as most studies were conducted in developed countries where farming practices are more intensive and control and/or eradication programmes have been implemented since decades. In Africa, most comprehensive epidemiological studies have been done in Zambia ,18,19,20In East Africa, pastoralists keep multiple species, mainly cattle, sheep, goats, camels and donkeys, often in large herd sizes. The grazing strategy in the area relies on the movement of livestock to follow grazing and water resources over considerable distances following seasonal changes. Most studies focusing on risk factors associated with pathogens that infect multiple host species involved single species and neglected the effect of multiple hosts . It is tPrevious studies have been carried out to investigate the roles that wildlife species play on the dynamics of bTB transmission ,29. WildMost studies focusing on ecological risk factors associated with pathogens that infect multiple host species tend to concentrate on industrialized countries, whereas the epidemiology of bTB in the developing world, especially in Africa, remains largely unknown . MoreoveThe study was carried out in Awash National Park and in the neighbouring Afar Region, Ethiopia. Study sites were selected based on a gradient of wildlife-livestock interactions, livestock production systems, concentrations of livestock and wildlife, and the presence of common grazing and water resources. In the southern tip of the region, most of the grazing land and watering points are shared by livestock and wild animals from the Awash National Park . It is vTragelaphus strepsiceros), which are known wildlife hosts of M. bovis [Awash National Park (ANP) is located in the Ethiopian Rift valley   with a sM. bovis ,38.2 [The Afar region is located in the north-eastern part of Ethiopia , and ‘sub-district’ (n = 34) as lowest sampling stages was used to select study villages. Study animals were obtained using a three-stage random sampling procedure. The ‘village’ within the sub-district was regarded as the primary unit, the ‘herd’ as secondary unit and ‘individual animal’ as tertiary unit. Herds of livestock in each sub-district were stratified into three groups based on herd size  after calculating the average herd size of the sub-district. Herds (one herd from each stratum) and individual animals were selected randomly. A total of 102 livestock herds from 34 sub-districts  were selected. Informed consent was sought verbally from all livestock herders and herd owners.The sample size was determined by assuming that the average expected prevalence of bTB was 11% . The desTuberculin skin testing was performed using aliquots of 0.1 ml of 2500 IU/ml bovine purified protein derivative (PPD) and 0.1 ml of 2500 IU/ml avian PPD . Bovine and avian PPDs were injected by veterinary staff intradermally at two sites approximately 12 centimetres apart at the border of the anterior and middle thirds of one side of the neck. This was done after shaving the two sites using a razor blade. The skin thickness was measured with digital callipers prior to and 72 h after PPD injection. An animal was considered bTB positive if the reaction at the bovine site minus the reaction at the avium site was greater than ≥ 4 mm cut-off, according to the recommendations of the World Animal Health Organization . In thisTo identify risk factors associated with bTB prevalence, semi-structured interviews with the herders and herd owners were conducted, gathering information on general herd management practices, livestock movement and transfer, introduction of animals into the herd, pastoral production system, other livestock species kept, types and levels of herd contacts, water sources (during the wet and dry season) and contact with wild animals . Local aThe interviewer estimated the maximum movement distance of the livestock herd by tracing the herd movement in the area based on interview-derived information, bound by roads, streams, rivers or hills, village, district, sub-district, or region, wildlife habitat or park, or other physical indicators, which were located on a georeferenced map. Subsequently, the maximum daily distance was calculated for sedentary and for the transhumance herds. For each herd, the total number of individuals introduced into the herd or transferred was estimated. It is likely that herds that graze close together have similar bTB prevalence, due to mixing of animals. At larger distances, herd bTB prevalence might be different, either higher or lower, due to spatial variation in bTB prevalence. To assess the impact of scale on the effect of livestock transfer on herd bTB prevalence, the effect of the number of animals introduced into the herd to strengthen social relationships was analysed separately for herds within and for herds outside the average daily herd movement radius of 7.3 km . LivestoThis study was approved by Haramaya University, Ethiopia (Reference number HUP14/559/15).Generalized Linear Mixed Models (GLMM) were used to examine the effects of predictors on the bTB infection probability for each animal using herd as random factor, with a binary response as a dependent variable (bTB positive/negative). Different approaches were used to study the strength and the relative importance of the risk factors on bTB prevalence at herd level. Prior to developing our candidate models for the herd-level analysis, one-by-one univariate analyses were performed to identify potential risk factors, using the bTB prevalence as dependent variable in a Generalized Linear Model (GLM) . The number of bTB positive as well as the numbers of bTB negative cattle were specified in a two vector response variable by combining two vectors into a single object as dependent variable, comprising the bTB positive and negative cattle in a herd. Predictor variables with p<0.25 were recognized as potential risk factors, and were subsequently used to construct multiple regression models. For highly correlated independent variables, only the one causing the largest change in the log-likelihood function was added to the final global model to avoid multi-collinearity, which was assessed by checking the variance inflation factors (VIFs); the final VIF-results confirmed the absence of collinearity among explanatory variables . In addition, to investigate the effect of wildlife-livestock interactions on the prevalence of bTB, we included the interaction term between herd size and contact with wildlife after including all main factors. From the global model, candidate models were selected using ΔAIC (< 3) and Akaike weights (w>0.05), with the best approximating candidate model having the highest w, as described in Burnham and Anderson . Model aThe individual animal prevalence of bTB was 5.5%, whereas the herd level prevalence was 46% (47 out of 102 herds).Risk of bTB infection increased with increasing age, as animals older than ten years had a significantly higher probability of bTB infection. There was also a strong association between having a poor body condition score and bTB infection, but sex, lactation status and reproductive status were not related with bTB status .Based on the results of the GLM analyses, seven out of 10 variables were identified as potential bTB risk factors, namely, herd size, the average herd movement in a day, number of animals introduced into the herd, number of animals transferred between herds, number of camels in a herd, pastoral production system, and contact with wildlife. These were all positively associated with bTB prevalence . HoweverSpearman's correlation matrix showed that herd movement and introduction of animals into the herd were strongly correlated with herd size and number of animals transferred, respectively r>0.7; , and theVariables included in the multiple linear regression analyses were herd size, number of animals transferred, number of camels, production system and contact with wildlife . All of The model summary showed that in all candidate models and in the global model, herd size, contact with wildlife and the interaction of herd size and contact with wildlife were positively correlated to bTB prevalence and always significantly, while the effect of pastoral production system, number of camels, and transfer of livestock were not significant in all candidate models .The results of model averaging showed that with increasing herd size and when the herd was in contact with wildlife the bTB prevalence increased . The modo = 0.9, p<0.001), through herd movement  compared to an animal introduced into the herd that was obtained from a transfer within the daily herd movement radius .The analysis of the impact of spatial scale on the effect of livestock transfer on herd bTB prevalence showed that introduction of animals into the herd from outside the average daily herd movement radius of 7.3 km  reduced The overall individual bTB prevalence was 5.5%, comparable with other results reported from Ethiopia ,10,34,54M. bovis [In line with previous studies ,56,57,58M. bovis .Similar to what Cleaveland et al.  and AmenK. leche, [T. strepsiceros, 5, 33), and African buffalo , greate caffer; ) are knon Africa ,18,56. Tn Africa ,61,62. Wn Africa . This laThe significant interaction between herd size and contact with wildlife in our study meant that the effect of contact with wildlife on the prevalence of bTB became larger when the herd size was reduced . HoweverThe transfer of livestock, within and between clans, is a common practice in the area to spread risks, especially during periods of drought and/or conflicts. We found that transfer of animals was not positively correlated with bTB prevalence. Introduction of an infected animal into a bTB free herd or area is one of the major risk factors for introducing the disease ,63,64. WTwo types of production and grazing system are practiced in the area, i.e., village resident herds with an agro-pastoral production system, and transhumant herds, following a pastoral production system. bTB prevalence in Uganda was higher in agro-pastoral production systems of Uganda . HoweverIn conclusion, this study identified that bTB prevalence increased with increasing age of cattle and with decreasing body condition at individual animal level. Herd size is an important risk factor contributing to the prevalence of bTB in cattle, because larger herds have the need to move more to look for more pastures and thus end up in the wildlife conservation area where the probability of contact with wildlife maintenance hosts harbouring bTB is higher. Thus, based on the study, it is impossible to indicate the contribution of wildlife species in the transmission of bTB to cattle, and the direction of the spread of the pathogen between wildlife and cattle. Findings from this study add useful epidemiological information regarding bTB infection at the livestock-wildlife interface in Ethiopia. In order to improve this understanding, further surveillance and research on the disease ecology, including habitat use among different wildlife species with cattle, migration ecology, and population monitoring are needed.S1 Fig(TIF)Click here for additional data file.S1 Questionnaire(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


After a 24-hour withdrawal of MPD, SHR did not present any alterations in [3H]-Dopamine Uptake, [3H]-SCH 23390 binding and cAMP production; nonetheless, after a 10-day MPD withdrawal, the results showed a significant increase of [3H]-Dopamine uptake, of the quantity of [3H]-SCH 23390 binding sites and of cAMP levels in these animals. Finally, SHR that underwent a 10-day MPD withdrawal and were challenged with cocaine (10 mg/kg i.p.) presented reduced [3H]-Dopamine uptake and increased cAMP production. Wistar rats were affected by the 10-day withdrawal of MPD in [3H]-dopamine uptake but not in cAMP accumulation; in addition, cocaine was unable to induce significant modifications in [3H]-dopamine uptake and in cAMP levels after the 10-day withdrawal of MPD. These results indicate a mechanism that could explain the high comorbidity between ADHD adolescent patients under methylphenidate treatment and substance abuse in adult life.Methylphenidate (MPD) is one of the most prescribed drugs for alleviating the symptoms of Attention Deficit/Hyperactivity Disorder (ADHD). However, changes in the molecular mechanisms related to MPD withdrawal and susceptibility to consumption of other psychostimulants in normal individuals or individuals with ADHD phenotype are not completely understood. The aims of the present study were: (i) to characterize the molecular differences in the prefrontal dopaminergic system of SHR and Wistar strains, (ii) to establish the neurochemical consequences of short- (24 hours) and long-term (10 days) MPD withdrawal after a subchronic treatment (30 days) with Ritalin® , (iii) to investigate the dopaminergic synaptic functionality after a cocaine challenge in adult MPD-withdrawn SHR and Wistar rats. Our results indicate that SHR rats present reduced [ Attention deficit/hyperactivity disorder (ADHD) is one of the most common childhood diseases, affecting approximately 3–7% of worldwide school-aged children. Although there is a tendency for symptoms to disappear over time, some individuals continue to present them at adulthood , 2. GeneThe prefrontal cortex (PFC) is involved with cognitive processes thought to comprise executive function, which includes impulse control, response inhibition, attention, working memory, cognitive flexibility, planning, judgment, and decision-making . Some ofSeveral reports describe Spontaneously Hypertensive Rats (SHR) as one of the most appropriate animal models to study ADHD since SHR are hyperactive and show defective sustained attention in behavioral tasks , 10. SHRDopamine transporter (DAT) activity is also believed to be critically involved in the dopaminergic dysfunction associated with ADHD and, therefore, should constitute a crucial molecular target for the treatment of this disease . SeveralMethylphenidate (MPD) is one of the most prescribed drugs for alleviating the symptoms of ADHD. The efficacy of this treatment has been widely described before . EvidencDespite the benefits of MPD treatment in ADHD, several reports express concern about the misuse of MPD associated with drug abuse , 21. It However, the molecular mechanisms of the dopaminergic system affected by MPD that could be involved in the susceptibility to other psychostimulants are not completely understood. So, in the present study we used SHR and Wistar rats to characterize dopamine uptake and cAMP production in both strains. We also evaluated dopaminergic changes in the PFC induced by chronic Ritalin® treatment (Methylphenidate Hydrocloride), followed by a short-term (24 hours) and a long-term (10 days) MPD withdrawal. Finally, we challenged SHR and Wistar rats with a single dose of cocaine in order to analyze the neurochemical effects of this psychostimulant on a PFC dopaminergic system previously exposed to MPD.All experiments were carried out under institutional approval of the Animal Care and Use Committee of the Universidade do Estado do Rio de Janeiro (CEUA/001/2013), in accordance with Brazilian Law n° 11.794/2008 and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. All efforts were done to minimize the number of animals used and their suffering.Spontaneously Hypertensive Rats (SHR) and Wistar rats were maintained in our institutional animal care facility in Universidade Federal Fluminense . Wistar rats represent an outbred heterogeneous control model for SHR for neurochemical analyzes. Original breeding stock was obtained from Universidade do Estado do Rio de Janeiro . SHR were mated within the same brood to ensure strain isogeny. Therefore male SHR from brood A1 would only mate with female from brood A1. However, to ensure Wistar heterogeneity, male rats only mated with female rats from different broods. Thereby, a male Wistar from brood B1 could never mate with a female from brood B1 and their descendants (B2 onwards). The day of birth was considered postnatal day 0 (PN0).°C) room on a 12-h light/dark cycle (lights on at 8:00 a.m.), with unlimited access to food and water.At weaning , male rats from each brood were separated from their progenitors and housed 2–4 per cage (49 × 34 × 16 cm polypropylene cage containing wood shavings) in a temperature-controlled (22±13H]-Dopamine (specific activity: 56.8 Ci/mmol) and [3H]-cAMP (specific activity: 28.1 Ci/mmol) were acquired from PerkinElmer . [3H]-SCH23390 (specific activity: 70.3 Ci/mmol) was acquired from Amersham (USA). All other chemicals were of the highest purity obtainable from regular commercial sources. Each experiment was performed at least 3 times.Methylphenidate hydrochloride  was produced by Novartis AG . Cocaine hydrochloride, R(+)-SCH 23390 hydrochloride (SCH 23390) and Dopamine Hydrochloride were purchased from Sigma Aldrich Co. . -Dopamine (1.2 x 10-6M) buffered to pH 7.4 with 20 mM HEPES at 37°C. The medium was removed and the tissue washed four times with 3 mL of cold Hanks 4 solution. This procedure was sufficient to wash out the free radioactivity (not taken up by the tissue). After the washes, 1 mL of Milli-Q water was added to the tissue to disrupt the cells. Following successive freeze-thaw cycles, cellular radioactivity was assayed using a scintillation counter. In a separate experiment, a time-course of [3H]-Dopamine uptake was performed at the following time points (in min): 10, 30, 60, 90 and 120.Samples of prefrontal cortex from SHR and Wistar rats were incubated for 60 min in 1 mL of MEM  containing 1 μCi -SCH 23390 binding assay protocol was carried out as previously described -SCH 23390 (70.3 Ci/mmol) in a final volume of 0.2 mL. (+)-SCH 23390 was added at the final concentration of 5 mM to determine nonspecific binding. Assay tubes were incubated at room temperature for 1 h and the reaction was terminated by rapid filtration through GF/C glass fiber filters pretreated with 0.3% polyethyleneimine. Radioactivity bound to the filters was quantified by liquid scintillation spectroscopy. Binding assays were performed in duplicate and in four different experiments.The [escribed . Briefly3H]-Dopamine uptake between naïve SHR and Wistar rats, unpaired Student t tests were used. The analysis of [3H]-Dopamine uptake at different time points in both strains was carried out by using a two-way ANOVA: Strain (SHR or Wistar) and Time Point  were used as between-subjects factors. Differences in cAMP accumulation between strains in a basal condition and after exogenous dopamine stimulation were also analyzed by a two-way ANOVA: Strain (SHR or Wistar) and Condition  were used as between-subjects factors. A one-way ANOVA was used to analyze the effect of MPD administration and withdrawal in SHR animals: Group  was considered the between-subjects factor. Two-way ANOVAs having Treatment (Control or MPD) and Duration of Withdrawal  as between-subjects factors were used for the following analyses: DAT expression, D1R expression and [3H]-SCH 23390 binding. Differences in cAMP accumulation among SHR groups as a function of MPD treatment, SCH use, and duration of withdrawal were also analyzed using a two-way ANOVA: Group  and Duration of Withdrawal (24 h or 10 d) were used as between-subjects factors. Finally differences between SHR and Wistar rats regarding [3H]-Dopamine uptake and cAMP accumulation as a function of MPD treatment and COC use were analyzed by two-way ANOVAs: Strain (Wistar and SHR) and Group  were used as between-subjects factors. Unpaired Student t tests and Fisher’s Protected Least Significant Difference (FPLSD) tests were used post hoc. For main effects and interactions, significance is assumed at the level of p < 0.05.Data are compiled as means ± S.E.M. The Kolmogorov–Smirnov one sample test (K–S) was used to assess the normality of the distributions of each of the variables. In order to compare [3H]-Dopamine uptake in slices of PFC showed a significant reduction of basal dopamine uptake of SHR in comparison to Wistar rats .As previously described, MPD  is a potent blocker of catecholamine reuptake . We haveprotocol . A signiTo determine whether MPD effects on [3H]-Dopamine uptake required changes in DAT or D1R expression in SHR animals, we performed a Western Immunoblotting assay . No diff3H]-SCH 23390 as the specific D1-like antagonist to verify whether specific D1-like receptor expression on the cell surface would be modified after MPD treatment  and cAMP accumulation . We therefore decided to analyze the data of each strain separately.SHR and Wistar rats treated with MPD for 30 days that underwent a 10-day withdrawal were submitted to a single administration of cocaine (10 mg/kg i.p.) 20 minutes prior to euthanasia (PN65) in order to investigate the neurochemical effects of cocaine in a system previously exposed to MPD: [3H]-Dopamine uptake in Wistar rats, a significant effect of group was observed  (Regarding [= 0.012) . This ef= 0.012) : cAMP le3H]-Dopamine uptake   . However< 0.001) . In this3H]-Dopamine uptake and D1-like-dependent cAMP accumulation in SHR but not in Wistar rats. Finally, we demonstrated that a single dose of cocaine is able to modulate the dopaminergic circuitry in SHR animals previously exposed to methylphenidate in a manner that differs from what was observed regarding Wistar rats.Here, we have shown that SHR animals present reduced dopamine uptake and basal cAMP production in comparison to Wistar rats. We have also shown that exogenous dopamine exposure or systemic administration of cocaine are not efficient in inducing neurochemical prefrontal responses in SHR animals. Furthermore, it was also demonstrated that a 10-day withdrawal from chronic methylphenidate treatment results in increased -Dopamine uptake and cAMP production were not reduced after a single administration of cocaine in VEH-treated SHR animals, Wistar neurochemical responses were strongly affected. Our data supports the hypothesis that the deficiency in the dopamine uptake system and the absence of response in the D1R-dependent cAMP production after dopaminergic stimulation in untreated SHR animals participate in the reduction of the functionality of the dopaminergic system that has been described in many studies. In addition to the low release of dopamine [We have also noticed that exogenous dopamine stimulation does not modify cAMP levels in naïve SHR animals, a result that markedly differs from what was observed in Wistar rats. Similarly, while [dopamine , a decreInterestingly, Gronier  emphasizMethylphenidate has been used as an effective treatment for ADHD for many years , 35. RepHarvey et al  demonstrThe absence of a significant methylphenidate effect after a 24-hour withdrawal on DAT activity does not seem to be the result of the use of an insufficient dose. For instance, we have shown that a single dose of methylphenidate (2.5 mg/kg p.o.) substantially reduces dopamine uptake in the prefrontal cortex of SHR animals, which is consistent with findings observed in the human brain . LikewisIt is well stablished that methylphenidate can act on the dopaminergic system and increase dopamine release , which, It is relevant to notice that we have observed an alteration in dopamine uptake but not in D1-dependent cAMP levels after long methylphenidate withdrawal in Wistar rats. Somkuwar et al  reportedThe consequences of chronic treatment with methylphenidate or its withdrawal on cocaine abuse are poorly understood. Several studies indicate that methylphenidate treatment during adolescence in ADHD individuals may increase substance abuse liability , 50, 51.We demonstrated that cocaine was ineffective in inducing an enhancement of cAMP levels in VEH-treated SHR animals, markedly differing from what was observed in Wistar rats, which presented a diminished uptake and increased cAMP levels, as expected. Furthermore, we have shown that a cocaine challenge enhances cAMP levels in a D1-like dependent mechanism in SHR pretreated with methylphenidate, while it reduced cAMP levels in methylphenidate-treated Wistar rats. It is possible that the neurochemical effects observed in SHR animals are a consequence of an association between the reinforcing mechanisms of methylphenidate and the inborn maladaptive function of the SHR dopaminergic system. Our findings support the results of previous studies: Harvey et al  have shoDespite the effects found by Beaudin et al. , which iIn conclusion, our study offers additional insight into the dopaminergic mechanisms mediating methylphenidate use during infancy and adolescence in animal models with or without the ADHD phenotype, and the neurochemical consequences at adulthood. Our results unveil functional aspects of chronic methylphenidate treatment in the prefrontal cortex of these adolescent animal models, and show how methylphenidate withdrawal causes changes in the dopaminergic system of this ADHD animal model. These findings indicate that SHR animals, after a prolonged withdrawal of methylphenidate, present increased dopaminergic sensitivity to psychostimulants, such as cocaine. This may be a possible mechanism that explains the high comorbidity between ADHD adolescent patients under methylphenidate treatment and substance abuse in adult life.S1 FigA significant group effect was observed  regarding cAMP accumulation, with the following rank order: MPD+SKF > MPD > SKF = Control. Results are expressed as means ± S.E.M. *** = p < 0.001, vs. Control. a = p < 0.01, b = p < 0.001, vs. SKF. c = p < 0.001, vs. MPD.(PDF)Click here for additional data file.S1 File(DOC)Click here for additional data file.S2 File(PDF)Click here for additional data file.


The hallmark of Parkinson Disease (PD) is the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the consequent striatal dopamine (DA) deficiency, although it is well recognized that neurodegeneration in PD goes beyond the SNc. Major advances have occurred in recent years on the molecular and pathophysiological basis of PD, however there remain many questions and unknowns regarding SNc cells vulnerability, and the exact significance of Lewy bodies and alpha-synuclein (α-syn) aggregation process regarding disease onset and progression. This Research Topic discuss the etiopathogenesis of PD, presenting a series of papers that provide up-to-date, state-of-the-art information on molecular and cellular mechanisms involved in the neurodegeneration process, neuroimmune pathways, the role of functional and anatomical organization of the basal ganglia as a factor of neuronal vulnerability, the possibility that PD is a prion disease and the cellular response to α-syn aggregation. Understanding the mechanisms underlying vulnerability of dopaminergic midbrain neurons and how pathology becomes widespread are primary objectives of basic and clinical research in PD.in vivo models of PD. Whereas PD can be sporadic, genetic or possibly related with toxic/infectious agents, a differential pattern of cell loss among midbrain dopaminergic neurons is observed regardless of disease etiology suggesting that differential dopaminergic neuron vulnerability does not depend on the factor triggering PD “per se” but on intrinsic properties of these specific cell groups. Here, Brichta and Greengard (Are dopaminergic and other neurons dying by the same pathogenic mechanisms? Do they all die to the same extent or at the same rate? What are the molecular determinants of susceptibility to the disease? To gain insights into these questions, researchers mainly rely in animal models. Blesa and Przedborski  provide reengard  providesreengard  clearly reengard  revealedAging is another major risk factor for developing PD. Rodriguez et al.  reviewedThe dopaminergic neurons of the SNc project primarily to the striatum, but also provide significant innervation of other basal ganglia nuclei and the thalamus. Villalba et al.  discuss Formation and accumulation of misfolded α-syn aggregates are a central and very hot topic of PD research currently. Several authors review and discuss here the importance of this protein, it's role in different cellular domains (Guardia-Laguarta et al., While all the features summarized above play a significant role in nigro-striatal neurodegeneration, it is unlikely that the origin of neurodegeneration in PD could be tight to a single pathogenic mechanism, hence the importance of defining markers and features of neuronal vulnerability. Obeso's group introduces here the interesting hypothesis that Parkinson's disease could be related and ultimately be the consequence of human multi-tasking behavior (Hernandez et al., In sum, understanding the mechanisms responsible for intrinsic SNc neuronal vulnerability is mandatory to progress in stopping neurodegeneration in PD. We trust that this Research Topic will spark new ideas and foster further advances in PD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cytokinesis is the final process in the cell cycle that physically divides one cell into two. In budding yeast, cytokinesis is driven by a contractile actomyosin ring (AMR) and the simultaneous formation of a primary septum, which serves as template for cell wall deposition. AMR assembly, constriction, primary septum formation and cell wall deposition are successive processes and tightly coupled to cell cycle progression to ensure the correct distribution of genetic material and cell organelles among the two rising cells prior to cell division. The role of the AMR in cytokinesis and the molecular mechanisms that drive AMR constriction and septation are the focus of current research. This review summarizes the recent progresses in our understanding of how budding yeast cells orchestrate the multitude of molecular mechanisms that control AMR driven cytokinesis in a spatio-temporal manner to achieve an error free cell division. The physical separation of the two daughter cells is called cytokinesis. Cytokinesis involves a periodic interplay between biochemical processes and cellular mechanics 2The core of the AMR is comprised of actin filaments, myosin-II heavy chain (Myo1), essential and regulatory myosin light chains (Mlc1 and Mlc2) and associated proteins, which coordinate AMR constriction with septum formation  through a process that involves Plk1 (polo-like kinase 1) mediated phosphorylation of the Rho1 GEFs Taken together, the assembly of the AMR is a complex process, which occurs stepwise throughout the cell cycle, but can be simplified to three key stages: (1) actin cable formation and stabilization at the cell division site by Rho1 activated formins, profilin and tropomyosin; (2) Iqg1 interaction with actin cables and contribution to actin ring formation and stabilization and (3) myosin light and heavy chains forming a template for Iqg1 mediated actin-ring formation.3It was originally believed that the AMR works like muscle-sarcomeres through a “filament-sliding” mechanism, in which myosin-II motors walk along anti-parallel organized actin filaments thereby generating the force that drives AMR constriction AMR constriction was shown to be under the control of the SCF(Grr1) ubiquitin ligase by promoting Hof1 degradation AMR constriction is a complex process and we are just beginning to understand the molecular mechanisms involved. The motor domain of Myo1, the regulatory myosin light chain Mlc2 and actin dynamics clearly contribute to AMR constriction. The molecular mechanism of the latter two and which role the primary septum plays remains to be fully elucidated.44.1N-actylglucosamine). Chitin synthases are integral membrane proteins with multiple trans-membrane domains. Budding yeast has three chitin synthases (Chs1–Chs3). Chs2 is the major enzyme responsible for primary septum formation, whereas Chs1 and Chs3 function in cell wall repair and bud scar formation via the secretory pathway to the cell division site AMR constriction is accompanied by centripetal formation of the primary septum , Fig. 3.4.2vice versa. This would have a strong impact on the interpretation of data surrounding AMR constriction in different mutants. Hence, mutations that affect primary septum formation could be wrongly interpreted as mutations that directly affect the contractility and force generation of the AMR. These circumstances make it difficult to interpret data about proteins whose functions in cytokinesis are not completely understood. Among these proteins are the F-BAR protein Hof1, the C2 domain containing protein Inn1 and the transglutaminase-like protein Cyk3. These proteins are known to interact with each other in vitro. The role of Cyk3 is less clear, as it is not essential for primary septum formation via the SH3 mediated interaction with Inn1 and Cyk3 AMR constriction goes hand in hand with primary septum formation. This raises the question as to whether both processes are interdependent and whether a defect in primary septum formation can impair AMR constriction and Cells without Myo1 can form a primary septum Taken together, efficient AMR constriction and primary septum formation are interdependent. It appears that the primary function of the AMR is to guide centripetal septum formation. On the other hand septum formation is important to stabilize AMR constriction during cytokinesis.4.3via its plasma membrane interacting C2 domain and an AMR interacting tail via a ring localization sequence (RLS) during AMR contraction. This domain was shown to form together with Iqg1 and Mlc1 a trimeric complex, which is part of the AMR via the septum promoting proteins (Hof1-Inn1-Cyk3) and Chs2 (via its SH3 domain with Hof1, Cyk3 and Inn1 To be able to guide primary septum formation, the AMR must be linked to the plasma membrane. Two ways of interaction could be imagined. The AMR could be either linked to the lipid bilayer, or to an integral or peripheral membrane binding protein. Inn1 was originally described to couple AMR constriction with furrow ingression and Chs2 . Interes5After AMR contraction, the secondary septum is deposited on both sides of the primary septum . The secTaken together, Rho1 is in addition to its function in AMR assembly the major regulator of secondary septum formation. This requires sequential Rho1 activation and inhibition steps in a spatio-temporal manner to allow the successive formation of the AMR and secondary septum and subsequent cell separation.6in vitro system that is able to simulate in vivo characteristics of the cell division machinery. This would allow one to investigate the underlying mechanisms in a fully tunable manner. In vivo evidence has demonstrated that the formation of extracellular matrix and the involved membrane associated proteins might be essential for the function of the AMR and even for its assembly. The implementation of these aspects in an in vitro system will be challenging. Nevertheless, recent progresses in in vitro systems have shown that many of the challenges can be addressed with a new array of tools Research over the last two decades has identified essential components of the cell division machinery as well as their complex interactions throughout the cell cycle. Structural information, investigations into the molecular mechanisms and of key and regulatory components have given insight into how the AMR drives cytokinesis. However, we do not completely understand the basic mechanisms that drive and coordinate AMR constriction and septum formation. A future milestone in the field will be to establish an


Osmunda is paraphyletic and needs to be separated into Osmundastrum and Osmunda s.str. Here, however, we describe an exquisitely preserved Jurassic Osmunda rhizome (O. pulchella sp. nov.) that combines diagnostic features of both Osmundastrum and Osmunda, calling molecular evidence for paraphyly into question. We assembled a new morphological matrix based on rhizome anatomy, and used network analyses to establish phylogenetic relationships between fossil and extant members of modern Osmundaceae. We re-analysed the original molecular data to evaluate root-placement support. Finally, we integrated morphological and molecular data-sets using the evolutionary placement algorithm.The classification of royal ferns (Osmundaceae) has long remained controversial. Recent molecular phylogenies indicate that Osmunda pulchella and five additional Jurassic rhizome species show anatomical character suites intermediate between Osmundastrum and Osmunda. Molecular evidence for paraphyly is ambiguous: a previously unrecognized signal from spacer sequences favours an alternative root placement that would resolve Osmunda s.l. as monophyletic. Our evolutionary placement analysis identifies fossil species as probable ancestral members of modern genera and subgenera, which accords with recent evidence from Bayesian dating.Osmunda pulchella is likely a precursor of the Osmundastrum lineage. The recently proposed root placement in Osmundaceae—based solely on molecular data—stems from possibly misinformative outgroup signals in rbcL and atpA genes. We conclude that the seemingly conflicting evidence from morphological, anatomical, molecular, and palaeontological data can instead be elegantly reconciled under the assumption that Osmunda is indeed monophyletic.The online version of this article (doi:10.1186/s12862-015-0400-7) contains supplementary material, which is available to authorized users. Osmunda L., Osmundastrum C.Presl, Leptopteris C.Presl, and Todea Bernh. This small group of ferns is remarkable in many respects and, consequently, has attracted considerable scholarly attention. Its members represent the most primitive of all leptosporangiate ferns  and in O. pluma C.N.Mill., O. iliaensis C.N.Mill., O. shimokawaensis M.Matsumoto & H.Nishida, and Todea tidwellii. Except for Todea tidwellii (placed at the root of either Leptopteris or Todea), all swaps occur within the Osmunda s.l. sub-tree. Swaps among the Jurassic species mostly involve placements at the root of the Plenasium sub-tree, the subgenus Osmunda sub-tree, and at the branch between Osmundastrum and the remaining Osmunda. Osmunda shimokawaensis and O. iliaensis are variably placed within the O. lancea Thunb.–O. japonica Thunb. sub-tree.Of all taxa placed via the evolutionary placement algorithm ; Ashicaulis (= Millerocaulis sensu Vera) claytoniites Y.M.Cheng and O. wehrii C.N.Mill. (at the root of the Plenasium sub-tree); O. arnoldii, O. bromeliifolia (C.Presl) Copel., and O. dowkeri ; O. oregonensis (C.A.Arnold) C.N.Mill. (at the root of subgenus Osmunda), and L. superba (at the branch of L. hymenophylloides).By contrast, fixed placements congruent over all three weighting schemes employed occur in: fossil members of et al.  to fully bipinnate and from fully to variably hemi-dimorphic.The systematic relationships revealed from our analysis of anatomical characters of the rhizomes reflect the distribution of gross morphological and fertile features within Osmundaceae very well. The isolated position and tight clustering of subgenus Todea and Leptopteris. These genera, having a rhizome structure broadly similar to that of Osmunda and especially Osmundastrum .An electronic supplementary data archive (ESA) containing all original data files and results, including the employed matrices in NEXUS format is available for anonymous download at


Introduction. Familial cases of Kaposi's sarcoma have rarely been reported. Kaposi's sarcoma is not uncommon in Greece; its incidence is estimated at 0.20 per 100.000 habitants, showing an increased predominance in the Peloponnese, in Southern Greece. Case Report. We describe five cases of familial clustering of KS originating from Greece. Discussion. The pathogenesis of familial Kaposi's sarcoma is still far from being completely understood. Genetic, environmental, and infectious factors have been incriminated. Kaposi's sarcoma (KS) is a multifocal disease that was first described in 1872 by Moritz Kaposi. It has four principal clinical variants: (1) classic KS, (2) African endemic KS, (3) KS in iatrogenically immunocompromised patients, and (4) AIDS-related epidemic KS. Classic KS presents as blue-red to violet macules on the distal lower extremities that coalesce to form large plaques or develop into nodules or polypoid tumors. Initially, unilateral lesions may progress to a more widely disseminated multifocal pattern. Early lesions may regress, while others evolve and lead to lesions at different stages. Patients may also have lesions in the mouth and gastrointestinal tract that are usually asymptomatic.Classic Kaposi's sarcoma mainly affects men over the age of 50 years, generally Jewish or of Mediterranean/Eastern Europe descent, and is also known as “Mediterranean KS.” The highest incidence is observed in the Mediterranean area, especially in Sardinia and Southern Italy . In ItalAlthough familial occurrence is rare, clustered occurrences within families have been reported in Sardinia and in the Peloponnese, in Southern Greece . FamiliaThis is a retrospective study that reports five families, each of which had two members affected by KS. The patients were HIV-negative and had no recognized underlying immunodeficiency . Four of6 IU, 3 times per week). After one year, the dose was reduced to 3 × 106 IU once per month. At the last follow-up visit, all lesions displayed a marked decrease in the size.A 78-year-old man, who originated from Southern Greece, was referred to “A. Sygros” Hospital exhibiting reddish-brown cutaneous lesions over different parts of his body that had manifested 18 years earlier. A biopsy specimen from lesional skin confirmed the clinical diagnosis of KS . The patA 36-year-old heterosexual man, son of the above mentioned patient, visited the outpatient clinic of “A. Sygros” Hospital with a red lesion that had appeared on his upper and lower extremities three years earlier. A skin biopsy indicated Kaposi's sarcoma . The pat6 IU three times per week). Partial remission was observed 6 months later.A 72-year-old woman was admitted to “A. Sygros” Hospital with multiple violaceous cutaneous lesions all over her body . The dis2. After 6 cycles, almost complete remission of the disease was achieved. Both patients came from the Peloponnese, Southern Greece.The son of the above patient, at the age of 51, was referred to “A. Sygros” Hospital with reddish cutaneous lesions in his left abdominal area. Biopsy specimens demonstrated Kaposi's sarcoma. Alitretinoin gel 0.1% was administered. The patient returned two years later with disseminated cutaneous KS. The patient was treated with doxorubicin pegylated 20 mg/mA 72-year-old female, who originated from Central Greece, was admitted to our hospital for treatment of recurrent Kaposi's sarcoma. The disease had started 3 years earlier, at the age of 69, with the appearance of a few violaceous macules and papules on the right lateral malleolus area. The lesions were diagnosed as KS and treated with radiotherapy. Six months before admission to our hospital, she displayed a recurrence of the disease. Localized radiotherapy resulted in remission of the lesions. At the scheduled follow-up visit, six months later, the lesions displayed noticeable improvement.Her sister, aged 51, visited the outpatient clinic of “A. Sygros” Hospital with reddish-purple lesions on her hands and on the legs . TopicalA 65-year-old man was referred to “A. Sygros” Hospital with reddish-brown cutaneous lesions over different parts of his body. The lesions had developed 8 months earlier, first appearing on his left thigh and gradually spreading to other parts of his body. The lesions were treated with cryosurgery. At the scheduled follow-up visit, three months later, the patient displayed a relapse and was treated with alitretinoin gel 0.1%. By the last follow-up visit, there was a marked decrease in five of seven lesions.6 IU, 3 times a week) for one year. After two months of follow-up, lesions were markedly decreased. Both patients originated from the Peloponnese, Southern Greece.His brother, aged 79, visited the outpatient clinic of “A. Sygros” Hospital with a few purple macules and papules on his lower extremities. Clinical examination revealed several reddish-purple macules and a biopsy indicated Kaposi's sarcoma. The patient was treated with subcutaneous recombinant interferon alpha-2a  or Kaposi's sarcoma-associated herpes virus (KSHV). The prevalence of KSHV varies among geographic regions from 2% to 7% in Western Europe and North America, from 10% to 20% in the Mediterranean, and up to 100% in Sub-Saharan African countries . KSHV inFamilial clustering of KS is rare and this fact argues against simple Mendelian inheritance. Nevertheless, the possibility that complex predisposing factors are involved cannot be excluded . We pres


Gliosarcoma, a variant of glioblastoma multiforme, is a dimorphic tumor known for its intra-axial occurrence and poor survival of less than a year. Here is an 11-year-old boy with gliosarcoma. He had a near total excision and postoperative chemoradiotherapy. He has lived through the disease for over 34 months with a residual disease.This case report is to report an unusual long survival of gliosarcoma in a teenager . Gliosarcoma is known for its rarity occurrence in older individuals and poor survival. It constitutes only 2% of aggressive astrocytomas or glioblastoma –4. Even An 11-year-old boy presented to the outpatient department with headache and vomiting in August 2011 [Hematoxylin and eosin stained tissue sections revealed a highly cellular neoplasm composed of predominantly spindle shaped cells with pleomorphic oval to elongated hyperchromatic nuclei . The celA differential diagnostic consideration of (1) gliosarcoma with leiomyosarcomatous differentiation and (2) teratoma with malignant transformation was made.Immunostaining for Vimentin showed cytoplasmic positivity in 70% of the cells. Smooth muscle actin showed focal weak positivity in 30% of the cells. Immunostaining for S100, epithelial membrane antigen, was found to be negative and glial fibrillary acidic protein was scanty positive; Ki67 proliferation index was 10–15% and P 53 protein was positive Figures , and 9. The patient was given external beam radiotherapy 66 Gy at 2 Gy in 33 fractions and 6 cycles of chemotherapy with temozolomide 200 mg per day for 5 days every 28 days . The posGliosarcoma or Feigin tumor, a variant of glioblastoma multiforme, constitutes 5–10% of gliomas . GliosarThe pathognomonic biphasic pattern of gliosarcoma constitutes the presence of gliomatous and sarcomatous components. There is also a theory of independently arising gliomatous and sarcomatous tumors called “collision tumor.” There is significant diagnostic dilemma which this entity poses on account of the gliomatous and mesenchymal components. The biphasic pattern is confirmed by reticulin fibers that highlight the reticulin-rich sarcoma component and glial fibrillary acidic protein (GFAP) positivity in immunohistochemistry, which demonstrates the glial component. Gliosarcomas also present with adenoid formation signifying metastatic carcinoma, with chondroid elements such as osseous metaplasia and with smooth muscle elements such as rhabdomyosarcomatous –15.The treatment for gliosarcoma is through multimodality approach. Complete resection followed by chemoradiation is a must. However most of the patients portend dismal prognosis , 17. LitPediatric gliosarcoma is evidently rare, and survival dictated by the disease is poor even with the best of multimodality approaches. This case of an 11 years old boy in his preadolescence treated with near total excision and chemoradiotherapy which has weathered the storm of the disease surviving to live his adolescence with good performance despite his residual disease is worth reporting.


Prolonged mesolimbic dopamine concentration changes have been detected during spatial navigation, but little is known about the conditions that engender this signaling profile or how it develops with learning. To address this, we monitored dopamine concentration changes in the nucleus accumbens core of rats throughout acquisition and performance of an instrumental action sequence task. Prolonged dopamine concentration changes were detected that ramped up as rats executed each action sequence and declined after earned reward collection. With learning, dopamine concentration began to rise increasingly earlier in the execution of the sequence and ultimately backpropagated away from stereotyped sequence actions, becoming only transiently elevated by the most distal and unexpected reward predictor. Action sequence-related dopamine signaling was reactivated in well-trained rats if they became disengaged in the task and in response to an unexpected change in the value, but not identity of the earned reward. Throughout training and test, dopamine signaling correlated with sequence performance. These results suggest that action sequences can engender a prolonged mode of dopamine signaling in the nucleus accumbens core and that such signaling relates to elements of the motivation underlying sequence execution and is dynamic with learning, overtraining and violations in reward expectation. Considerable evidence suggests that midbrain dopamine cell activity1235789121314It was suggested that prolonged dopamine concentration changes could provide the sustained motivational drive required to navigate through space to distal rewards, because, among other findings, dopamine concentration scaled flexibly with distance to reward1617On the basis of our previous between-subject evaluation of mesolimbic dopamine signaling during execution of an instrumental sequencei.e., free-operant) action-sequence task devoid of experimenter-provided initiation cues. Subjects controlled both the initiation of each sequence and the speed with which it was performed. The initiating lever was continuously available and when pressed resulted in the insertion of the terminating lever into the chamber. The terminating lever remained out until it was pressed, at which point 0.1 ml of an orange-flavored 12.5% sucrose solution reward was delivered and this lever was retracted. Levers were located on the wall opposite the food-delivery port to dissociate dopamine signals related to collection of the earned reward from those related to initial approach towards the instrumental manipulandum and action execution. Rats earned 30 total rewards/session.Fast-scan cyclic voltammetry (FSCV) was used to measure dopamine concentration changes in the NAc  of food-6,60 = 8.52, p = 0.001; th training session, from which point on sequence completion time was lower than the first day of training (p < 0.05–0.01) and improved no further (p > 0.05). The first day of sequence training was, therefore, considered initial acquisition, with training sessions 2 and 3 considered ‘pre-asymptotic performance’, 4 and 5 considered ‘at asymptotic performance’, and 6 and 7 considered ‘extended training’ beyond asymptotic performance. With training, rats also began to re-initiate the next action sequence after earned reward collection more quickly .Rats’ behavior improved over the 7 days of action-sequence training . The estimated time of the initial approach was identified in video recordings synchronized to the voltammetric data scored to determine when prior to the press the lever came into the rat’s sight line and then reversing the tape to identify the earliest movement on this path. Because the self-paced task allowed variable sequence times, dopamine concentration v. time traces were concatenated by scaling the peri-event windows to the lowest mean inter-event interval between consecutive events  and plotting the data from each event to half of the inter-event interval, similar to the method described previouslyAs is apparent in the single-trial, representative examples  and group < 0.01) over the entire action sequence period, as described previouslyp < 0.01) linear regression coefficient was detected in 10/11 rats (r170 = −0.60–0.85), which was positive for the majority (7/11) of rats , and a significant negative correlation between average dopamine elevation onset time and training session .NAc dopamine concentration began to rise increasingly earlier in the execution of the action sequence with training . There w2,20 = 5.40, p = 0.01) and of Training , and an interaction between these factors . Post-hoc analyses  nor Trial block × Session phase interaction  on dopamine signaling during any training session.The dopamine response peak time also appeared to shift with training. During initial acquisition dopamine peaked during reward delivery/consumption following sequence termination, but with training the peak occurred earlier in the execution of the action sequence and at asymptotic performance occurred prior to execution of the initiating action. These observations were confirmed by statistical analysis of the maximal dopamine concentration change during sequence execution across training epochs  did not significantly differ between these sessions, ruling out this movement variable as an explanation for the disappearance of the dopamine response with extended training. These data suggest that NAc dopamine became unrelated to reward delivery and individual action sequence performance during extended training.After extended training peak dopamine concentration amplitude preceding sequence initiation did not significantly correlate with sequence speed , with no effect of Type of Path , but a significant interaction between these factors . Although dopamine was not significantly elevated above baseline when rats executed the sequence on the overtrained stereotyped path , it was significantly elevated above baseline if they deviated from this path . Moreover, from the time of initial approach through reward delivery following sequence termination dopamine concentration was significantly lower than that elicited at asymptotic performance if the action was the overtrained stereotypical path (p < 0.05–0.001), but not if it was the overtrained atypical path (p > 0.05).Performance during extended training became largely stereotyped, but there were rare instances in which rats deviated from this stereotyped path, paused, or otherwise became disengaged in the task. We took advantage of these atypical trials to ask whether NAc dopamine signaling would be differentially engaged when rats reengaged in the task. Following reward collection, rats could to turn to the left or right upon exiting the food-delivery port to move to the opposite wall to reinitiate another sequence. By extended training rats developed a stereotypical path (path chosen >70% of the time). Atypical path trials  occurred when rats deviated from their stereotyped path , paused on the stereotyped path before action execution, or otherwise became disengaged in the task. As can be seen in the representative examples  and grou4,38 = 4.35, p = 0.006 on peak dopamine concentration, Because NAc dopamine signaling became decoupled from individual, stereotyped sequence performance after extended training, we next asked if this signal transitioned to an earlier task element. We evaluated dopamine signaling surrounding the most distal and unpredictable, task cue- that signaling the start of the session . Secondarily, this analysis provided an opportunity to verify dopamine detection throughout training. The magnitude of dopamine response to this start cue was minimal early in training and became maximal on the last training session  we performed a series of preference tests in a separate group of rats. In the first test the relative palatability of each reward was evaluated by measuring lick frequency, total licks and pauses in licking (lasting > 0.5 s) during isolated, non-contingent deliveries of each reward in 30, 0.1 ml increments, during separate daily sessions22232,11 = 95.04, p < 0.0001; 3,21 = 18.00, p < 0.0001; post hoc p < 0.0001, in all cases; 7 = 3.19, p = 0.02), but following the taste aversion rats did consume less of this higher value reward, even though it had not itself been paired with illness , indicating that it shared many specific identifying features with the orange-flavored 12.5% sucrose.In this separate group of subjects, we also verified that each alternate identity reward was discriminable from the orange-flavored 12.5% sucrose. We conditioned a taste aversion to the orange-flavored 12.5% sucrose, by pairing with LiCl-induced nausea, and then repeated the consumption choice tests. The taste aversion treatment was effective; rats consumed significantly less of the orange-flavored 12.5% sucrose after it was paired with illness , with neither an effect of Sequence event , nor a significant interaction between these factors . Although there was evidence of sustained dopamine elevations during sequence execution, there was no clear evidence of the ramping characteristic in the dopamine signal during this value expectation violation test. The linear regression coefficient ranged from −0.51–0.68, with 33% of subjects showing a significantly positive slope and 50% showing a significantly negative slope on the trial-averaged dopamine concentration v. time trace.For each expectation violation test in the FSCV experiment we first confirmed dopamine detection elicited by the session start stimulus. For the value expectation violation test this stimulus elicited a significant dopamine response and the magnitude of this response did not differ between test and the preceding training control session . Apparent differences in the magnitude of the start cue-evoked dopamine are due to between-subject variability in this response. In no case did the magnitude of the start cue dopamine response at test differ from that induced on the last training session day . Importantly, sequence execution-related dopamine signaling did not differ between any of the identity violation tests and their preceding control training session This finding is, however, inconsistent with the prolonged NAc dopamine signals detected during spatial navigation in presumably well-trained ratsAlthough NAc dopamine concentration was no longer elevated during stereotyped action sequence performance following overtraining, dopamine elevations were robustly elicited in response to the unexpected presentation of the most distal reward predictor, the session start stimulus. This backpropagation of the dopamine signal is consistent with recent findings of dopamine responses during performance of an action-sequence task largely similar to that used here, with the critical exception that it was on an experimenter-defined trial structure in which rats were unable to predict the time of the next sequence trial. Following post-asymptotic performance in this task, NAc core dopamine was transiently elevated by the unexpected presentation of the initiating leverNAc dopamine signaling was found to be reengaged after overtraining if the earned reward was unexpectedly larger in value, but not if only its specific identity  was altered. This suggests that NAc dopamine  encodes information about the motivational value, rather than the specific sensory features of rewards. This interpretation is consistent with previous work demonstrating NAc dopamine responses to valuable sucrose reward in the absence of its identifying sweet taste4546phasic dopamine signaling in reporting the reward-prediction errors vital for associative encoding123643135051The prolonged dopamine signals detected here during action sequence execution and previouslyFindings inconsistent with prediction error encoding by prolonged dopamine elevations do not suggest refutation of this theory, but rather highlight the possibility that there are multiple roles for dopamine, some over different time scales4955125560et al. (2013), prolonged dopamine elevations may reflect the sustained motivational drive required to pursue distal rewards. In support of this here, dopamine elevations preceding sequence initiation predicted the speed of the immediately following sequence, corroborating our previous report63186566676869707374As proposed by Howe Male, Sprague Dawley rats  were group housed and handled daily prior to surgery for 5–7 days. Rats were maintained on a food-deprived schedule whereby they received 10–12 g of their maintenance diet daily in order to maintain approximately 85% free-feeding body weight. Rats were provided free access to tap water in the home cage and were fed approximately 3–4 hr after each daily training session. All procedures were conducted in accordance with the NIH Guide for the Care and use of Laboratory Animals and approved by the UCLA Institutional Animal Care and Use Committee.Carbon-fiber microelectrode preparation and calibration procedures are identical to those described previously19Training commenced between 30–36 days post-surgery . An orange-flavored 20% sucrose reward was substituted  for the reward value expectation violation. During a pre-training consumption test (15-min access to each reward on successive days) rats consumed more of this reward than the orange-flavored 12.5% sucrose training outcome  to confirm the relative value and discriminability of each violation test reward against the orange-flavored 12.5% sucrose that served as the earned reward in the main experiment. Two preference tests were used. In the first, the relative preference for each reward was tested in consumption choice test wherein each violation reward was pitted against the orange-flavored 12.5% sucrose. On separate days rats were given 15-min access to the orange-flavored 12.5% sucrose along with one of the violation test rewards . All tests were conducted 18 hr food deprived, unless the alternate water reward was used, in which case rats were 0 hr food deprived, but 18 hr water deprived. In the second, test the relative palatability of each reward was evaluated by measuring lick frequency, total licks, and pauses of >0.5 s in licking during isolated non-contingent delivery of 0.1 ml of each reward (each reward delivered in separate test sessions) as described previouslyp < 0.05. Data were analyzed with one- and two-way ANOVAs (Geisser-Greenhouse correction), paired t-tests, correlation and regression where appropriate. Post-hoc comparisons correcting for multiple comparisons were used to clarify all main effects and interactions.Unless otherwise mentioned all data were processed with Microsoft Excel  and MATLAB . Statistical analyses were conducted with GraphPad Prism  and SPSS . For all hypothesis tests, the α level for significance was set to Action-sequence time (time between the initiating lever press and reward collection) was the primary behavioral output measure. Re-initiation time (time from reward collection to the next initiating lever press) was used as a measure of the self-imposed inter-trial interval. The slope of the change in average sequence time from the first training session to asymptotic performance was the measure of learning rate.Video recordings of all experiments were synchronized to behavioral and electrochemical data and were scored to identify the time at which rats began movement towards the initiating lever. Time of this ‘initial approach’ was estimated by determining when prior to the press the lever came into the rat’s sight line  and then reversing the tape to identify the earliest movement on this path. Only those initiating movements that culminated in an actual lever press were included in this analysis.or a pause in movement or movement away from the lever on the typical path, such that when rats re-engaged in the sequence they did so from a different starting location. One rat was omitted from this analysis due to an inability to score typical v. atypical path.Stereotyped v. non-stereotyped response paths during the extended training phase were also evaluated in these recordings. When rats earned a reward they could either turn to the left or to the right upon exiting the food-delivery port to make the initiating movement to the back of the box to reinitiate another sequence and were free to pause or otherwise deviate from the direct path to the lever. By extended training rats developed a stereotypical path, defined as the path that chosen >70% of the time . An atypical path was defined as either the alternate to the typical path  in vitro from a sample of representative carbon fiber microelectrodes, as has been described previously11306364in vivo background drift from these electrodes were also including in the training set, as previous work has dictatedin vitro pre-test calibration factor.Electrochemical data were analyzed using software written in LabVIEW . Principal component regression (PCR), a chemometric technique that combines principal component analysis with inverse least-squares regression77Voltammetric data were isolated and concatenated starting 5 s before the initial approach toward the initiating lever and ending 5 s after the delivery of the earned reward. Concatenation was performed by scaling the peri-event windows to the lowest mean inter-event interval between consecutive events  and plotting the data from each event to half of the inter-event interval, similar to the method described previouslyin vivo FSCV dataQ) from the PCR analysis were compared to the average tolerance level (Qα), which in these measurements was 174.12 Q/nA2. On occasion isolated transient electrical artifacts were detected, as identified by transient Q values well in excess of the Qα in isolated cells. Estimates of dopamine at these time points were interpolated by taking the average of the surrounding cells. Trials in which >10% of the data points had residual values in excess of the Qα were omitted from the analysis. This occurred on <5% of all trials. All PCR residual results are provided in Average sequence time was always considerably less than the 90-s limit on PCR prediction of Voltammetric data for the test phase were analyzed similarly with the exception that the initiating press, rather than initial approach served as the first analyzed event to focus on reward- and action-related dopamine concentration changes. Data were also analyzed for the period 5 s prior to and 10 s after the start of the session . Peak dopamine concentration change was quantified for each trial in each analysis window and this value was averaged across each training/test session for each rat. Root mean square (RMS) noise in similar time epochs outside of the behavioral sessions  are provided as a reference for this measure.How to cite this article: Collins, A. L. et al. Dynamic mesolimbic dopamine signaling during action sequence learning and expectation violation. Sci. Rep.6, 20231; doi: 10.1038/srep20231 (2016).