Starting with a protein structure or binding site, ProBiS-ligands first identifies template proteins in the Protein Data Bank that share similar binding sites. Based on the superimpositions of the query protein and the similar binding sites found, the server then transposes the ligand structures from those sites to the query protein. Such ligand prediction supports many activities, e.g. drug repurposing. The ProBiS-ligands web server, an extension of the ProBiS web server, is open and free to all users at  The number of protein structures in the Protein Data Bank (PDB) is increasing rapidly  patterns by comparing the query protein with all the protein chains in the nr-PDB using ProBiS algorithm  in the 3D space using a fast density clustering algorithm with a distance cutoff of 5 Å for proteins and nucleic acids, and 3 Å for small molecules and ion ligands . PredictLigand 3D Viewer and on the right side the Ligand Tabs  to structurally conserved (red), as in the ProBiS web server  and the predicted ligands (CPK colored sticks) are visualized in an integrated JSmol molecular viewer . This vib server . The useSmall Molecules, Proteins, Nucleic Acids or the Ions tab opens the corresponding interactive table, one for each ligand type; a table for small-molecule ligands is shown in Figure View 3D link in the Ligand column shows the predicted ligand in the Ligand 3D viewer, and zooms in on the ligand; the View 3D link in the BSite column shows invariant binding site residues for the corresponding ligand. The Name column has the names of the ligands, and Structure column shows a small picture of the 2D ligand structures that is enlarged when a mouse cursor is over it.On the right side of the output page, clicking on the Glyoxalase family protein shown in panel A has no sequence homologues in the PDB; however, ProBiS-ligands predicts three ion clusters based on the detected invariant residues in various distantly related glyoxalases. In panel B, the query protein (3tkz) is an SH2 domain and the predicted ligand is a protein transposed from the source PDB (1r1s) which shares ∼30% sequence identity with the query protein. This protein–protein complex is not seen in 3tkz and similar protein structures, and the possibility of its existence probably has been overlooked previously. Panel C shows a binding site in bacterial enzyme D-alanine:D-alanine ligase with an endogenous ATP ligand and transposed inhibitor of biotin carboxylase (<30% sequence identity). This enables one to find different fragments of inhibitors bind to the same location in a query binding site, which could be used to design new compounds as a combination of existing ligands, i.e. ligand homology modeling. Finally, in panel D, a predicted DNA ligand bound to endonuclease IV query protein is shown. In ProBiS-ligands, we currently do not remove steric clashes between the predicted ligands and the query proteins; we expect to address this in the future.ProBiS-ligands can be used to construct protein–ligand complexes Figure  by transZ-scored predicted specific ligand from the first small molecule or ion clusters with the actual known ligands of the query proteins using an in-house developed 2D molecular graph matching algorithm. Ligand similarities, expressed as Tanimoto coefficients, were averaged over the predictions, and range between 0 and 1, where 1 is the highest similarity.We assessed the performance of the ProBiS-ligands web server on 500 protein models and their corresponding experimental structures Table . This tehttp://probis.cmm.ki.si/ligands/benchmark.ProBiS-ligands predictions are better for experimental protein structures than for protein models, which suggests that the ProBiS algorithm is relatively sensitive to the structural accuracy of query proteins. To simulate the lack of similar templates, a situation that occurs frequently in protein structures from structural genomics projects, we consecutively excluded template proteins sharing >30%, >20% and >10% sequence identity with the query proteins from the test set. The performance dropped when similar templates were unavailable; however, for experimental query structures, reasonable predictions with MCC of 0.28 and ligand similarity of 0.40 were possible even when templates with <10% sequence identity were available; for protein models, templates with at least 20–30% sequence identity were required for similar prediction accuracy. Our results show that ProBiS-ligands predicts ligand clusters that correlate well with actual ligand binding sites even when only evolutionary unrelated templates are available. The benchmark results can be found at ProBiS-ligands is a web server for prediction of ligands based on detected local structural similarities in proteins. One of the major advantages of ProBiS-ligands is that it allows transposition of ligands between protein structures irrespective of protein folding and with no prior knowledge of binding sites. This allows an established ligand, e.g. a drug, to be seen in a new perspective in which binding to other proteins, not hitherto recognized as targets, can be recognized. Repurposing of established drugs follows directly from this. We envision that the construction of accurate models of known ligands in binding sites will enable design of more specific ligands.


Microstructural inhomogeneity generally deteriorates the corrosion resistance of materials due to the galvanic effect and interfacial issues. However, the situation may change for nanostructured materials. This article reports our studies on the corrosion behavior of SiC nanoparticle-reinforced Al6061 matrix composite. It was observed that the corrosion resistance of Al6061 increased when SiC nanoparticles were added. Overall electron work function (EWF) of the Al-SiC nanocomposite increased, along with an increase in the corrosion potential. The electron localization function of the Al-SiC nanocomposite was calculated and the results revealed that valence electrons were localized in the region of SiC-Al interface, resulting in an increase in the overall work function and thus building a higher barrier to hinder electrons in the nano-composite to participate in corrosion reactions. Microstructural inhomogeneity generally deteriorates the corrosion resistance of materials due to the galvanic effect and interfacial issues. The difference in electrochemical behavior between different phases may accelerate corrosion reactions, leading to higher susceptibility to electrochemical attacks. For metal-matrix composites in which the reinforcing phases are usually non-conductive ceramic materials, the interfacial properties become crucial.The interface between the matrix and reinforcement plays an important role in property control for composites3589In general, the chemical reactivity of materials could be evaluated by their polarizability, which is a property for measuring how the material responds to the external field1214Electron work function (EWF), as an important physical property to evaluate the driving force for the electron flow, has been widely applied in microelectronicsSiC nanoparticles  were co-milled with nitrogen gas atomized Al-6061 powder (38–63 μm) in a laboratory planetary ball mill (PM-2400). The mixed powers contained 1, 2 and 3 vol% SiC nanoparticles, respectively. The nominal chemical composition of Al6061 is given in 2SO4 solutions was evaluated through electrochemical polarization measurement. Before the polarization test, each specimen connected to a copper wire was mounted in epoxy resin with a surface area of 1 cm2 exposed to the corrosive solution. The exposed surface was ground with silicon carbide papers up to 800-grit and cleaned with acetone. A saturated calomel electrode (SCE) was used as the reference electrode and a platinum plate with area of 1 cm2 was used as the counter electrode. The corrosion current density was determined using the Tafel extrapolation method.The corrosion behavior of the samples respectively in 3% NaCl and 0.1 mol/l H−8 Hartree. As for the ELF, it is defined as2627The first-principle method was employed to calculate work function of (111) plane of Al and (100) plane of silicon carbide (SiC), and the electron localization function (ELF) of valence electrons in interfacial zone between Al and SiC. The interface was constructed by connecting (111) plane of Al with (100) plane of SiC, followed by relaxation of the system to minimize the lattice misfit energy. The calculations were implemented with an ABINIT package22whereandρ is the first-order reduced density matrix with spin dependence, where 3C4, were observed in the nanocomposite. From the SEM image, one may see that SiC nanoparticles were embedded in the Al matrix in a satisfied manner, no aggregation of SiC nanoparticles was observed on the surface. Furthermore, the TEM image also shows that the SiC nanoparticles are homogeneously distributed in the Al mixture and the particle size ranges from 20 nm to 60 nm.In 2SO4 solutions, respectively, are presented in 2SO4 solution is a strong corrosive medium and can corrode various metals in a uniform corrosion mode. In contrast, in the NaCl solution, Cl− ions play a main role in corrosion, usually resulting in pitting and intergranular corrosionPolarization curves, corrosion potential and corrosion rates of Al-SiC nanocomposites with different volume fractions of SiC nanoparticles in 3 wt.% NaCl and 0.1M HThe damage to a material by corrosion is affected by two determining factors, one is corrosion rate that is related to corrosion kinetics, and another is the corrosion tendency or potential. The lowered corrosion rate of Al6061 with SiC nanoparticles, compared to that of Al6061, could be partially ascribed to the fact that the SiC particulates basically remained inert in the corrosive solutions, which would not promote local galvanic effect, and the dispersed SiC particles reduced the metallic surface area that was exposed to the solution, thus leading to lowered corrosion rate.Another factor is the corrosion tendency, which is represented by the corrosion potential. A higher corrosion potential represents a lower tendency for corrosion. As shown in ϕm) and the electron work function (φm) of the metal can be generally expressed asThe increase in the corrosion potential generally results from lowered activity of valence electrons that participate in corrosion reactions, which is reflected by raised electron work function (EWF). In order to see whether or not the EWF had the capability of monitoring the variation in activity of valence electrons with the interface density in nanocomposites, EWFs of the nanocomposites was measured and are illustrated in ϕm of a metal varies due to different values of where In order to understand the mechanism for the increase in the overall EWF with the added SiC nanoparticles, we calculated electron work functions of Al (111) crystal plane and SiC (100) plane. Selecting these planes for the calculation is attributed to the fact that the low-index planes with low surface energies are thermodynamically stable and more representative. The calculation shows that electron work functions of Al (111) crystal plane and SiC (100) plane are 4.01 ev and 5.16 eV, respectively see . When a Considering the rise in EWF is an indication of an increase in valence electron densityet al.An important issue here that needs to be clarified is “by which means the SiC nanoparticles can increase the EWF of the nanocomposites”. For a metallic material, its overall EWF is usually increased by adding elements with higher EWF if the added elements are dissolved in the material without forming second phases2SO4 solutions, respectively, were investigated. It was observed that the corrosion resistance of the Al-SiC nanocomposite was higher than the Al matrix (Al6061). Corresponding electron work function (EWF) of the Al-SiC nanocomposite increased, accompanied with an increase in the corrosion potential. The first-principle calculation indicates that the improvement in corrosion resistance is attributed to the electron localization in the interfacial region between the Al matrix and SiC particle. Such an effect would become profound when the SiC particle size is reduced to the nanoscale, with which the interfacial area is significantly increased along with markedly reduced spacing between adjacent SiC nanopartiles, leading to considerably enhanced electron confinement.In summary, the corrosion behavior of SiC nanoparticle-reinforced Al6061 matrix composite in 3% NaCl and 0.1M HHow to cite this article: Mosleh-Shirazi, S. et al. Interfacial valence electron localization and the corrosion resistance of Al-SiC nanocomposite. Sci. Rep.5, 18154; doi: 10.1038/srep18154 (2015).


The retrosplenial cortex supports navigation, but there are good reasons to suppose that the retrosplenial cortex has a very different role in spatial memory from that of the hippocampus and anterior thalamic nuclei. For example, retrosplenial lesions appear to have little or no effect on standard tests of spatial alternation. To examine these differences, the current study sought to determine whether the retrosplenial cortex is important for just one spatial cue type  or whether the retrosplenial cortex helps the animal switch between competing spatial strategies or competing cue types. Using T-maze alternation, retrosplenial lesion rats were challenged with situations in which the available spatial information between the sample and test phases was changed, so taxing the interaction between different cue types. Clear lesion deficits emerged when intra- and extra-maze cues were placed in conflict (by rotating the maze between the sample and choice phases), or when the animals were tested in the dark in a double-maze. Finally, temporary inactivation of the retrosplenial cortex by muscimol infusions resulted in a striking deficit on standard T-maze alternation, indicating that, over time, other sites may be able to compensate for the loss of the retrosplenial cortex. This pattern of results is consistent with the impoverished use of both allocentric and directional information, exacerbated by an impaired ability to switch between different cue types. The retrosplenial cortex (areas 29 and 30) comprises the posterior part of the rodent cingulate cortex and is thought to contribute to spatial cognition as well as contextual and episodic memory . There aDespite the foregoing evidence, there are reasons to suppose that the retrosplenial cortex has a very different role in spatial memory from that of the hippocampus and anterior thalamic nuclei. This potential functional difference is highlighted by the outcome of permanent lesions in these three sites on T-maze alternation. In this task, a rat typically runs up an alley and is then forced into one side arm (‘sample’). After a delay, the rat runs up the alley and is rewarded for alternating, i.e., selecting the opposite arm to that entered in the sample phase (‘choice’). Both hippocampal and anterior thalamic lesions produce very clear deficits, even when the task is made easy by having short retention periods and by extending the inter-trial intervals to reduce interference . In contThe present study examined several different explanations for the seemingly mild effects of retrosplenial cortex lesions. One explanation centers on the spatial demands of the task. Alternation in a T-maze could be based on multiple cues types, e.g., allocentric, intra-maze, and directional . If retrA second explanation arises from the hypothesis that the retrosplenial cortex helps the animal switch between competing strategies or competing cue types . In normA agonist muscimol. Previous studies that have made transient retrosplenial lesions by infusing tetracaine suggest that this approach might be more disruptive to spatial learning than permanent lesions (A third explanation (Experiment 3) is that other sites, e.g., the hippocampus and anterior thalamic nuclei, are able to compensate, over time, for loss of the retrosplenial cortex. Such a mechanism would help explain the mild deficits associated with permanent retrosplenial lesions. For this reason, we sought to inactivate the retrosplenial cortex temporarily by infusing the GABA lesions , 2001.Subjects were male rats  weighing 222–262 grams at the time of surgery. Animals were housed in pairs under diurnal light conditions (14 h light/10 h dark) and testing was carried out during the light phase. Animals were given free access to water throughout the study. The animals were handled daily for at least 1 week before surgery. In Experiments 1 and 2 the rats were randomly assigned to one of two surgical groups: receiving ‘complete’ retrosplenial cortex lesions (‘RSC’) or sham operations (‘Sham’). A total of 15 (Experiment 1) and 14 (Experiment 2) rats received retrosplenial lesions, while 11 (Experiment 1) and 12 (Experiment 2) rats received sham surgeries. Separate cohorts of rats were used in Experiments 1 and 2. In Experiment 3, 12 new rats were implanted with guide cannulae into the retrosplenial cortex. Of these 12, eight were tested on the T-maze task and the remaining four used to image the spread of muscimol. All experiments were carried out in accordance with UK Animals (Scientific Procedures) Act, 1986 and EU directive 2010/63/EU.2). The rats were each placed in a stereotaxic headholder  with the nose bar at +5.0. The scalp was then cut and retracted to expose the skull. A craniotomy exposed the dura above the dorsal cortex. The lesions were made by injecting a solution of 0.09M N-methyl-D-aspartic acid  dissolved in phosphate buffer (pH 7.2).Animals were deeply anesthetized by an intraperitoneal (i.p.) injection (60 mg/kg) of 6% sodium pentobarbital . Anesthesia was maintained with isoflurane  at an infusion rate of 0.1 μl/min. The needle was left in place for 5 min after each infusion. The anterior–posterior (AP) coordinates were measured (in mm) with respect to bregma, the medio-lateral (ML) coordinates (in mm) with respect to the sagittal sinus, and the dorso-ventral (DV) coordinates (in mm) with respect to the surface of the cortex. The stereotaxic co-ordinates of the seven injections were: (1) 0.26 μl at -1.8 (AP), ±0.4 (ML), -1.0 (DV); (2) 0.26 μl at -2.8 (AP), ±0.4 (ML), -1.1 (DV); (3) 0.26 μl at -4.0 (AP), ±0.4 (ML), -1.1 (DV); (4) 0.26 μl at -5.3 (AP), ±0.4 (ML), -2.4 (DV); (5) 0.26 μl at -5.3 (AP), ±0.9 (ML), -1.4 (DV); (6) 0.26 μl at -5.3 (AP), ±0.9 (ML), -1.8 (DV); (7) 0.1 μl at -7.5 (AP), ±1.0 (ML), -1.1 (DV).On completion of the surgery, the skin was sutured and Clindamycin anti-biotic powder  was applied topically. Subcutaneous Metecam  provided peri-operative analgesia. Animals also received subcutaneous injections of glucose saline (5 ml). The surgical operated controls (‘Sham’) received the same procedure and drugs as the lesioned animals, with the exception of lowering the needle into the brain and injection of NMDA. Following surgery, rats were allowed a minimum of 2-weeks of post-operative recovery before behavioral testing.2 and maintained thereafter with isoflurane (1–2%). Stereotaxic surgery was conducted with the incisor bar set at -3.3 mm below the intra-aural line, so that the skull was flat. Given the rostral-caudal extent of the retrosplenial cortex, the rats were implanted with two bilateral guide cannulae: one targeting the rostral (approximately -2.5 posterior to bregma) and a second targeting the caudal (approximately -6.0 posterior to bregma) retrosplenial cortex. At approximately -2.5 (AP) from bregma a small flap of bone was removed to reveal the central sinus. Medial lateral coordinates were then taken with reference to the central sinus. At the rostral site, one bilateral stainless guide cannulae  was implanted ±0.7 from the central sinus. At the caudal site, two holes were drilled ±1.0 from the central sinus and a second bilateral stainless guide cannula  was implanted. Cannulae were held in place by dental cement and anchored to the skull with six fixing screws  located on the parietal bone plates. Removable obturators  were inserted into the guide cannulae to prevent the cannulae from blocking.Anesthesia was induced by isoflurane (4%) in OAll other aspects of the surgical procedures were the same as described for Experiments 1 and 2, except that the rats were allowed 1 week of post-operative recovery before behavioral testing.In Experiment 1 and 3, as well as stages 1–2 of Experiment 2, testing was conducted in a modifiable four-arm (cross-shaped) maze. The four arms  were made of wood while the walls (17 cm high) were made of clear Perspex. At any time, one of the arms could be blocked off to form a T-shaped maze. Aluminum barriers could be positioned ∼25 cm from the end of each arm to create a start area. For all experiments, the location of the start arm remained constant such that the T-maze was in the same orientation throughout testing. The maze, which was supported by two stands (94 cm high), was situated in a rectangular room (280 cm × 280 cm× 210 cm) with salient visual cues located on the walls.In stage 3 of Experiment 2, two identical cross-mazes were used. The mazes were placed side-by-side (with the end of the two side arms touching each other) on a table (74 cm high) in the same experimental room. The floor of each cross-maze was made from wood and painted white. Each arm was 45.5 cm long and 12.0 cm wide. The sidewalls (32.5 cm high) were made from opaque black Perspex. At the end of each arm was a sunken food well (2 cm in diameter and 0.75 cm deep). Access to an arm could be prevented by placing an aluminum barrier at the entrance of the arm.Lighting during initial habituation and training in the ‘light’ stages was provided by two standard ceiling lights, giving a mean light intensity of 140.8 lx (measured at the center of the maze). During training in the ‘dark’ stages, illumination was provided by two infra-red spotlights, with the experimenter using night-vision goggles (Productive Firm Dipol Ltd.) in order to see. Mean light intensity within the T-maze during training in the dark was less than 1 lx.One week prior to the start of the experiment, the animals were placed on a food restricted diet and gradually reduced to 85% of their free-feeding weights. One day before the start of the experiment, the rats were familiarized with the sucrose reward pellets  in their home cage.Pre-training began with 4 days of habituation to the maze. On Day 1, four sucrose pellets were scattered down each of the arms and two in each food well, and the rats were allowed to explore the maze for 5 min. On Day 1 only, rats were habituated in cage pairs. On Days 2 and 3, the number of pellets was increased to four in each well and the rats were habituated to the maze for 5 min. On Day 4, the number of pellets was reduced to two in each well and the aluminum barrier was placed at the entrance of one of the arms. The rats were again allowed to explore the maze for 5 min. During each habituation session, the reward pellets were continuously replaced so that no arm was found to be empty on return. Training on the forced-choice alternation rule began the following day.Figure 1). Although all testing was carried out in a cross-maze, it was modified by placing a barrier blocking access to the arm directly in line with the start arm (effectively turning it into a T-maze configuration).Throughout Experiment 1, the rats received six trials per daily session. In Experiments 2 and 3 this was increased to eight trials. Each trial consisted of a forced ‘sample’ run followed by a ‘choice’ run . The rat received two reward pellets only if it turned in the direction opposite to that in the sample run . The choice of the sample arm (left or right) was randomly assigned with the only stipulation that no arm could be selected as the sample on more than two consecutive trials. In Stage 3 of Experiment 2 (performed in the two adjacent cross-mazes) the sample phase was conducted in one cross-maze and the choice phase in the adjacent cross-maze.During the forced sample run, one of the side arms of the maze was blocked by an aluminum barrier. After the rat turned into the preselected arm, it was allowed to eat two reward pellets that had been previously placed in the food well. The rat was then picked up from the maze and immediately returned to the start arm. Approximately 10 s after the end of the sample phase, the choice phase began. The rat was allowed to run up the stem of the maze and was now given a free choice between the left and the right turn arms .Experiments 1 and 2 examined whether the involvement of the retrosplenial cortex in alternation problems is restricted to certain classes of cues. In order to control systematically the strategies supporting alternation, the alternation training consisted of different stages and trial types that allowed the availability of different cues to be selectively manipulated .In Stage 1 (six sessions), both the sample and choice run were carried out in the light in the single maze, with no rotation of the maze between sample and choice . In order to prevent the rats from acquiring a rule, the direction of rotation (either clockwise or anti-clockwise) was randomly selected so that on half the trials it was rotated clockwise and on the other half it was rotated anticlockwise (there were never more than two successive rotations in the same direction). There were two sessions  of standard trials followed by 4 days of rotation between the sample and choice phases, then finally 2 days of standard trials.In order to place intra-maze cues in conflict, the maze was rotated 90° between the sample and choice phases. Once the sample phase was completed, the rat was removed from the sample arm and the start arm was rotated 90° . There were 10 days of training with six trials per day . There then followed 2 days  in which both the maze was rotated and the lighting was switched off between the sample and choice phases .In order to probe the flexible use of the various cues available to the rats, the illumination in the room was changed between the sample and choice phases in some trials of Stage 3. The sample phase was conducted in the light but once the sample phase was completed, the lights in the room were switched off to preclude the use of distal visual cues . To exclude the use of allocentric cues, the remaining trials were conducted in the dark, with the maze again rotated between sample and choice .The rats had 16 days  in the single T-maze. Half of the trials on each day were conducted in the light, with the maze rotated between sample and choice , 2 days in the light with rotation , 4 days in the dark without rotation , and finally 2 days in the dark with rotation .In order to determine whether the within session switch in trial type may have contributed to the pattern of results obtained, the animals underwent a further 14 days of training  in which the trial type remained constant within each session. Rats had 4 days in the light without rotation . The sample phase was conducted in one maze and the choice phase in the adjacent maze. There followed 8 days of the same training except that it was conducted in the dark .The final stage was conducted in two adjacent T-mazes, so that any intra-maze cues were uninformative. Initially, the rats had 2 days (1 day in each maze) in a single maze with the sample and choice phases conducted in the same maze. Thereafter, the rats had 8 days  in the two mazes conducted in the light , the rats underwent 6 days of training on the standard T-maze task, receiving eight trials a day. This training was conducted in exactly the same manner as described for Stage 1 training in Experiments 1 and 2 and in the same adapted cross-maze.Following recovery from surgery, the rats were given 1 day of reminder training on the standard T-maze task. This training proceeded in exactly same manner as the pre-surgery training.The animals underwent two test sessions. Prior to these test sessions, the animals received either infusions of muscimol or phosphate buffered saline (PBS). Half the animals were tested first under muscimol and then tested after the control infusion. For the other half of the animals, the test order was reversed. An interval of 48 h separated each test.in situ for a further 1 min to allow absorption of the bolus. The infusion cannulae were then removed and the obturators replaced. The animals were returned to their home cage. After 30 min, the animals received eight standard T-maze trials.Muscimol  was dissolved in sterile PBS at a dose of 1 mg/ml . Rats weThe boundaries and nomenclature for the retrosplenial cortex are taken from In order to image the spread of muscimol around the injection site, four rats were infused with fluorophore-conjugated muscimol  into the retrosplenial cortex . The infp < 0.05. Simple effects were used to explore statistically significant interactions. To assess whether performance remained above chance  one sample t-tests were applied to the data, where appropriate.The number of correct responses per session and per trial type was noted. The percentage correct per trial type was then calculated and averaged across two sessions to give blocks of 12 (Experiment 1) and 16 (Experiment 2) trials. This measure was subjected to ANOVA with between subject factor group (RSC and Sham) and within subject factors of trial type and block. In Experiment 3, test performance was subjected to ANOVA with a within subject factor of infusion (muscimol and control). To confirm that the assumptions of ANOVA were met, the data were subjected to Levene’s Test of Equality of Variance, the Shapiro–Wilk Test of Normality and, where appropriate, Mauchely’s Test of Sphericity. The alpha level was set at Figure 2). In these cases, much of the cortical tissue had completely collapsed, but in other areas where tissue was still intact, the remaining cells looked abnormal and there was extensive gliosis. Anterior to the splenium, the lesions were essentially complete. Caudal to the splenium, there was partial sparing of granular a retrosplenial cortex in six cases  and in one case there was bilateral dysgranular sparing. Additional cell loss occurred in a discrete part of the most dorsal medial portion of CA1 in the septal hippocampus , seven cases unilateral . There was evidence of ventricular dilation in five cases (Experiment 1).A total of seven rats (three from Experiment 1 and four from Experiment 2) were excluded from all subsequent analysis, as these cases had either excessive sparing throughout the retrosplenial cortex or extensive bilateral hippocampal and subicular damage. In the remaining cases (12 in Experiment 1 and 10 in Experiment 2), the surgeries consistently produced marked cell loss throughout almost the entire retrosplenial cortex . As is clear from this figure, the RSC group were initially mildly impaired relative to the Sham group, but by the end of training there were no differences between the groups. ANOVA revealed an effect of lesion  but no effect of block or interaction between lesion and block . Performance in the RSC group was also significantly above chance levels across the three blocks of training .Figure 1B) led to a numerical drop in performance in both groups of animals . There was no statistical evidence that the two groups were differentially sensitive to maze rotation as there was an effect of rotation  and lesion  but no rotation by lesion interaction .Placing intra-maze cues in conflict , there was no difference in accuracy between the two groups  and performance in both groups was above chance for each of the five training blocks of trials .When all cues types were available , the RSC group appeared less sensitive to the intra-trial change in lighting conditions as performance in Sham animals dropped to around chance levels . But, as training proceeded, Sham performance recovered whereas the RSC group showed little or no improvement. ANOVA revealed an effect of block , a marginal block by lesion interaction  and no effect of lesion (F < 1). The RSC group showed no improvement in performance across the blocks of training (F < 1) but in the Sham group there was an effect of block  as accuracy improved across the five blocks of training. Further analysis showed that performance in the Shams was not above chance levels on blocks 1–2 (maximum t < 1) but was above chance on blocks 3–5 . In the RSC group, performance was above chance levels on blocks 1 and 5  but did not differ from chance on blocks 2–4 .Initially during the concurrent light/dark trials  neither group performed above chance levels . This failure to solve the problem was confirmed by one sample t-tests . ANOVA confirmed there was no difference between the two groups on these trials (F < 1).When the intra-trial change in lighting conditions was combined with maze rotation  appeared to selectively disrupt performance in the RSC group. In contrast, with the absence of visual cues , both groups appeared equally sensitive to the manipulation. ANOVA confirmed this description of the data as there was a main effect of lighting , lesion  but also a lesion by lighting interaction  but no other effects or interactions reached significance . The lesion by lighting interaction arose because there was an effect of lesion during light  but not dark (F < 1) trials.Inspection of t(11) = 3.6, p < 0.01] but in the dark it was only above chance on blocks 3–4 . Performance by the RSC group was above chance in the light on blocks 1–3  but in the dark it was only above chance on block 4 .Subsequent analysis confirmed that Sham performance in the light was consistently above chance , a marginal effect of rotation , an effect of lesion  and a marginal lesion by lighting by rotation interaction , but no other interactions . Subsequent simple effects analysis confirmed that there was an effect of lesion in the light + rotation condition  but not in the light without rotation condition . Conversely in the dark, Shams were more accurate than the RSC group , but when the intra-maze cues were placed in conflict both groups performed at equivalent levels . Further analyses revealed that performance by both the Sham  and the RSC group  was above chance for each of the four trial types.In Stage 2 there were four trials types  but trial type remained constant across each session  = 2.5, p = 0.13]. Mean percentage correct (±SEM): Sham = 86.4 (±3.3); RSC = 83.3 (±3.7).During pre-training (two sessions) in the adjacent cross-mazes (sample and choice phases run in the same maze) there was no difference in accuracy between the two lesion groups , an effect of lesion  but no other effects or interactions . The three-way interaction arose because in the light there were no effects of lesion or interaction between lesion and block . However, in the dark, there was a lesion by block interaction  as Sham, but not RSC, performance improved across the four blocks of training. Overall, the Shams were more accurate than the RSC group in the dark .To exclude the use of intra-maze cues, the sample and choice phases were thereafter run in separate mazes. The animals were tested in the light  = 5.9, p < 0.05] and was consistently above chance levels across all three blocks .All animals readily acquired the alternation problem. Performance improved across the three blocks of two sessions, as confirmed by an effect of Block . ANOVA, comparing mean performance before and after surgery, revealed no effect of cannulae implantation on performance .Following recovery from surgery, performance was again above chance . While performance under PBS was reliably above chance , performance was at chance following temporary inactivation of the retrosplenial cortex .Temporary inactivation of the retrosplenial cortex with muscimol severely disrupted performance on the standard T-maze task . The same intra-trial delay (10 s) was used across all trial types and the maze rotation occurred once the rat had been removed from the sample arm. Thus, the sensitivity to maze rotation was not due to any greater disruption of the animals or to longer retention periods. These selective lesion effects on intra-trial maze rotation are also consistent with studies using the eight-arm radial arm maze. Here, rats with retrosplenial lesions often appear unaffected during normal training, but when the maze is rotated after four correct arm choices (placing intra- and extra- maze cues in conflict), performance is excessively disrupted , presumably reflecting their excessive reliance on non-visual intra-maze cues, which could still guide spatial alternation. Other evidence concerning the relative use of distal visual cues comes from Morris water-maze studies, which show that retrosplenial lesions can impair place learning, whether acquired by actively swimming to an escape platform  is that rats with retrosplenial lesions are less likely to use distal allocentric or directional cues. The retrosplenial cortex, and in particular the dysgranular subregion, is densely connected with cortical areas  that provide visual information , consistrm e.g.,  or by merm e.g., . Furtherrm e.g., .Figure 8).Despite these consistent findings, the present study also shows that difficulties with using distal visual cues does not provide a full account of the effects of retrosplenial cortex lesions. One example concerns those alternation trials with a sample in the light and a choice trial in the dark . Despite an initial advantage, the rats with retrosplenial cortex lesions failed to improve over successive trials, even though a reliance on intra-maze cues should provide an effective strategy. Another inconsistency comes from Experiment 2 stage 3, which used two adjacent cross-mazes, one for the sample and one for the choice test. Now, there was little or no evidence of a retrosplenial lesion impairment when animals were tested in two adjacent mazes in the light, despite the apparent need to rely on distal visual cues . Even so, a lesion impairment still emerged when testing in the two mazes occurred in the dark. Clearly, the availability of distal visual cues is not a sufficient condition to produce a retrosplenial cortex lesion deficit. Instead, the pattern of results suggests that in the absence of intra-maze cues in the light, retrosplenial lesion animals are able to use other sources of spatial information  to support spatial alternation, but when forced to rely on directional information in the dark, a retrosplenial lesion impairment emerges . Similarly, when the trial type remained constant across sessions and the value of both allocentric and intra-maze cues was nullified in the dark with maze rotation , performance in the retrosplenial group remained above chance levels. As animals are unable to use egocentric response strategies to alternate  may arise from the extent to which animals appreciate the use of two rather than one maze, each in different locations. It is also possible that Sham rats are more aware of this change, e.g., because of greater sensitivity to different locations within the test room . Evidenc emerges . These dlternate , this lelternate , the finlternate . Furtherlternate . The redlternate , with alFigure 6).The inference is that the disruptive effects of maze rotation on retrosplenial performance are not solely due to a failure to use distal visual cues effectively. This conclusion leads to the second explanation, that the retrosplenial cortex helps the animal switch between competing strategies, e.g., competing cue types. With this explanation in mind, it is helpful to appreciate that maze rotation does not simply negate the value of intra-maze cues, it places them in conflict with other available cue types. To perform the T-maze task effectively under these circumstances, animals must presumably ignore the conflicting information and rely on those cue types most reliably associated with correct performance. The finding that T-maze performance in the Sham animals was barely affected by maze rotation in the light suggests that normal animals are rapidly able to disregard conflicting intra-maze cues and rely more on extra-maze cues to support effective spatial alternation. Consistent with this suggestion, Sham group performance dropped to levels observed in the retrosplenial lesion group when the maze was rotated in the dark and the use of allocentric cues was explicitly excluded, i.e., both intra- and extra- maze cues were removed  might suggest that intact animals are not always able to switch spontaneously between strategies, at least on a trial by trial basis . In both experiments, the animals were also subject to changes in cue type . Nevertheless, as training progressed, performance in the Sham animals recovered and was consistently above chance. In contrast, there was no equivalent improvement in the RSC animals. Thus, Sham animals were clearly able to adopt other available strategies  to support spatial alternation. The lack of improvement in the retrosplenial cortex lesion animals is indicative of a less flexible use of the various, available sources of spatial information.A agonist that spares axons, the current study rules out effects on fibers of passage. The present deficit shows how the importance of the retrosplenial cortex for spatial alternation can be masked by compensatory mechanisms, reflecting the parallel involvement of closely related structures. One prediction that follows is that this deficit should abate with repeated testing under muscimol as this testing regime would allow functional compensation by other structures to emerge. This compensation presumably involves the use of other spatial metrics such as intra-maze cues to solve the alternation task. As such, the results of Experiment 3 are also consistent with the notion that the retrosplenial cortex aids the flexible use of multiple spatial strategies. If, as would be predicted on the basis of the lesion data, temporarily silencing the retrosplenial cortex disrupts the use of allocentric and directional information, the animal is forced to rely on other classes of spatial information. Thus, one explanation for the profound effects of retrosplenial inactivation on alternation performance is that it compounds the deficit in allocentric and directional information by preventing the animal from switching to alternative strategies.A third explanation for seemingly intact T-maze alternation levels after retrosplenial cortex lesions presumes that compensatory mechanism in other structures partially mask retrosplenial involvement. We, therefore, temporarily inactivated the retrosplenial cortex with muscimol. In contrast to the effects of permanent lesions, temporary inactivation of the retrosplenial cortex resulted in a profound T-maze alternation deficit, comparable to that found after hippocampal or anterior thalamic nuclei damage . This apThe current study had three main goals. The first was to determine whether the retrosplenial cortex is important for just one cue type and the second goal was to test the specific hypothesis that the retrosplenial cortex helps the animal switch between competing strategies or competing cue types. One clear finding was that retrosplenial lesions do not preclude all spatial strategies. Consistent deficits only emerged when animals were forced to rely on directional information in the dark or when allocentric and intra-maze cues were placed in conflict. As has been found previously , these iThe 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.


Eptesicus fuscus). Our results reveal that both DTNs and non-DTNs can have responses selective to binaural stimulation, with a majority of IC neurons showing some type of ILD selectivity, fewer cells showing ITD selectivity, and a number of neurons showing both ILD and ITD selectivity. This study provides the first demonstration that the temporally selective responses of DTNs from the vertebrate auditory midbrain can be selective to binaural cues used for sound localization in addition to having spiking responses that are selective for stimulus frequency, amplitude, and duration.Electrophysiological studies on duration-tuned neurons (DTNs) from the mammalian auditory midbrain have typically evoked spiking responses from these cells using monaural or free-field acoustic stimulation focused on the contralateral ear, with fewer studies devoted to examining the electrophysiological properties of duration tuning using binaural stimulation. Because the inferior colliculus (IC) receives convergent inputs from lower brainstem auditory nuclei that process sounds from each ear, many midbrain neurons have responses shaped by binaural interactions and are selective to binaural cues important for sound localization. In this study, we used dichotic stimulation to vary interaural level difference (ILD) and interaural time difference (ITD) acoustic cues and explore the binaural interactions and response properties of DTNs and non-DTNs from the IC of the big brown bat ( Neurons throughout the auditory pathway respond selectively to the frequency and amplitude of an auditory stimulus. There is also a class of neurons first found in the auditory midbrain that have responses selective for a third acoustic parameter—signal duration—and these cells are called duration-tuned neurons (DTNs). In mammals, DTNs were first discovered from the inferior colliculus (IC) of echolocating bats  stimulation with sounds presented to the contralateral ear  and interaural time difference (ITD). When sound originates from a location directly in front of a listener , the received amplitude of the signal should be equal in both ears. Similarly, the path length and propagation time required for sound energy to travel to each ear should also be equal. When a sound source moves away from 0° azimuth and to the left of the auditory midline, the stimulus reaching the left ear will be larger in amplitude and will be received at a shorter time delay compared to the signal received by the right ear. The case is opposite for a sound source located to the right of midline. Interaural amplitude and time of arrival differences for sounds received by the two ears are important acoustic cues that the CNS uses to compute sound source location. Binaural ILD and ITD cues are first processed in the superior olivary complex within the auditory brainstem. In mammals, ITD cues are first processed in the medial superior olive  were selective to binaural ILD and ITD cues, thus establishing if their responses could possibly play a role in sound localization. Second, we wanted to compare the response properties and binaural selectivity of DTNs and non-DTNs to determine if these two neural subpopulations could be providing the CNS with different types of information relevant for sound localization. According to the duplex theory of sound localization, ILD cues dominate the localization of high-frequency sounds whereas ITD cues dominate the localization of low-frequency sounds . For the surgery, bats were first placed in a 12 × 10 × 10 cm anaesthesia induction chamber where they inhaled a 1 to 5% isofluorane:oxygen mixture (flow rate: 1 L/min). Anesthetized bats were then placed in a foam-lined body restraint within a stereotaxic alignment system that had a custom gas mask for continuous anesthetic inhalation (David Kopf Instruments Model 1900). The hair covering the skull was shaved and the overlying skin was disinfected with Betadine® surgical scrub. Local anesthetic  was injected subcutaneously prior to making a midline incision in the scalp. The temporal muscles were reflected, the skull was scraped clean and swabbed with ethanol, and the headpost was glued to the skull overlying the cortex with cyanoacrylate superglue (Henkel Loctite Corporation) cured with liquid acrylic hardener . One end of a chlorided silver wire that was attached to the headpost was placed under the temporal muscles and served as the reference electrode. Recordings began 1–4 days after surgery and each bat was used in 1 to 8 sessions lasting ca. 6 to 8 h. Recordings were terminated if a bat showed signs of discomfort. Between sessions, the electrode penetration site was covered with a piece of contact lens and Gelfoam coated in Polysporin®. Bats were housed individually in a temperature- and humidity-controlled room. All procedures were approved by the McMaster University Animal Research Ethics Board and were in accordance with the Canadian Council on Animal Care.Electrophysiological recordings were obtained from male and female cf = 7 kHz; high-pass cf = 300 Hz). Spike times were logged on a computer by passing the PC1 output to a spike discriminator (TDT SD1) and an event timer (TDT ET1) synchronized to a timing generator (TDT TG6).Electrophysiological recordings were conducted inside a double-walled, sound attenuating booth with electrical shielding . Prior to recording, each bat was given a subcutaneous injection of a neuroleptic . Bats were then placed in a foam-lined body restraint that was suspended by springs within a small animal stereotaxic frame that was customized for bats (ASI Instruments) and mounted atop of an air vibration table (TMC Migro-g). The bat's head was immobilized by securing the headpost to a stainless steel rod attached to a manipulator (ASI Instruments) mounted on the stereotaxic frame. The dorsal surface of the IC was exposed for recording by making a small hole in the skull and dura mater with a scalpel and a sharpened wire. Single-unit extracellular recordings were made with thin-wall borosilicate glass microelectrodes with a capillary filament  and filled with 3M NaCl. Electrode resistances typically ranged from 10 to 30 MΩ. Electrodes were positioned over the dorsal surface of the IC with manual manipulators (ASI Instruments) and were advanced into the brain with a stepping hydraulic micropositioner (Kopf Model 2650). Action potentials were recorded with a Neuroprobe amplifier (A-M Systems Model 1600) whose 10x output was bandpass filtered and further amplified (500 to 1000×) by a Tucker Davis Technologies spike pre-conditioner , programmable attenuators (TDT PA5) and signal mixers (TDT SM5) with equal weighting. The output of each mixer was fed to a manual attenuator (Leader LAT-45) before final amplification (Krohn-Hite Model 7500). Unless testing for binaural responses, stimuli were presented monaurally, contralateral to the IC being recorded, using a Brüel & Kjær o incidence; grid off) connected to a measuring amplifier (B&K Type 2606) and bandpass filter (K-H Model 3500), was quantified with a sound calibrator (B&K Type 4231) and expressed as decibels sound pressure level (dB SPL re 20 μPa) equivalent to the peak amplitude of continuous tones of the same frequency. The loudspeaker transfer function was flat ±6 dB from ca. 28 to 118 kHz, and there was at least 30 dB attenuation at the ear opposite the source , acoustic spiking threshold (dB SPL), and for DTNs we also determined the best duration (BD) and duration tuning response class  electrophysiological response characteristics of a cell, we then used binaural (dichotic) stimulation to systematically vary ILD and ITD acoustic cues to explore and compare the response properties of DTNs and non-DTNs. For DTNs the signal duration was set to the cell's BD, whereas for non-DTNs the signal duration was set to the same duration that was used to measure the neuron's BEF (see above). To measure the ILD response function of a cell, the SPL of the BEF signal presented to the contralateral ear was held at +10 dB (re threshold) while the SPL of the BEF signal presented to the ipsilateral ear was randomly varied in 5 dB steps ranging from −30 to +30 dB . Upon collecting these data, the SPL of the contralateral stimulus was increased to +20 dB (re threshold) and the evoked responses were recollected over the same range of ILDs. To measure the ITD response function of a cell, the SPL of the BEF signals presented to the contralateral and ipsilateral ears were equal and set to +10 dB  while the onset time of the signal presented to the ipsilateral ear was randomly varied in 10 μs steps from −250 to 250 μs (re contra stimulus). Upon collecting these data, the SPL of the binaural stimulus was increased to +20 dB (re contra threshold) and the evoked responses were recollected over the same range of ITDs. To facilitate the direct comparison of spiking responses evoked by different cells to varying ILD/ITD cues, spike counts were normalized by dividing the observed spike count at each ILD/ITD step by the maximum spike count evoked by the cell during ILD/ITD testing.For neurons with monotonic or near-monotonic ILD/ITD response functions, we fitted a four-parameter sigmoid function in a manner similar to Tollin et al.  and Karcx is the ILD or ITD value, A is the minimum asymptote, B is the slope factor, C is the inflection point, and D is the maximum asymptote. Using the fitted curves, we measured and compared the ILD50 or ITD50 point  in five bats and its value was 12.08 ± 0.84 mm. Maximum ITDs occur for sounds broadcast at 90° away from the auditory midline  and can be calculated with the formula:The maximum value for the biologically relevant range of ITDs in d is the interaural distance and c is the speed of sound. Assuming c = 344 m/s and given d = 12.08 ± 0.84 mm, this results in a maximum ITD of 35.12 ± 2.45 μs.where Maximum ITD values increase if the Woodworth formula is used to calculate the time required for sound to travel around a spherical head:r is the spherical radius, c is the speed of sound, and θ is the azimuthal sound source angle  between DTNs and non-DTNs tested with binaural stimuli at two suprathreshold levels. All statistical analyses were performed in SPSS or Python (SciPy) and used an experiment-wise error rate of α = 0.05.Unless stated otherwise, all data are reported as the mean ± standard error (SE). The proportion of DTNs and non-DTNs with ILD- and ITD-selective response functions at +10 or +20 dB above threshold were compared with a Fisher's exact test. Two-sample t-tests were used to compare basic electrophysiological response parameters  had responses that were selective for the duration of an acoustic stimulus, while 27 (48%) were not duration selective  because their spike counts remained at or above the 50% response maximum across all stimulus durations tested .The population of 29 DTNs was divided into three categories based on the duration filter (tuning) characteristic of the cell. Bandpass DTNs respond maximally at their best duration (BD) and have spiking responses that decline to ≤50% of the response maximum at stimulus durations both longer and shorter than BD. Shortpass DTNs are similar to bandpass neurons in that they also respond maximally at BD; however, their spiking responses only drop to ≤50% of the maximum at stimulus durations longer but not shorter than BD. Longpass DTNs do not have a BD and spike only when the stimulus duration meets or exceeds some minimum duration. Unlike typical energy integrating sensory neurons, both the number of spikes and first-spike latencies of longpass DTNs do not increase or decrease, respectively, with increasing SPLs  spiking with 19 responding to stimulus onset, 0 to stimulus offset, and 2 showing mixed spiking responding to both the onset and offset of the stimulus. The remaining 6 cells were classified as sustained responding because they had spikes evoked throughout the duration of the stimulus.vs. the stimulus duration of non-DTNs), BEF, and acoustic threshold. Within the population of cells tested, the mean BEF of DTNs was significantly higher than the mean BEF of non-DTNs; however, there were no significant differences in recording electrode depth, duration of the stimulus used to evoke a response, or acoustic threshold between DTNs and non-DTNs  response in every neuron. Presentation of a stimulus to the ear ipsilateral to the IC that was being recorded evoked either an excitatory (E), an inhibitory (I), or no response (O). Of 56 neurons, 36  were characterized as EI because they were excited by stimulation of the contralateral ear and inhibited by stimulation of the ipsilateral ear, 12  as EE because they were excited by stimulation of either ear, 7  as EO because they were excited by stimulation of the contralateral ear and neither excited nor inhibited by stimulation of the ipsilateral ear, and 1 cell (shortpass DTN) was unclassified because we did not characterize its binaural responses in this manner.dotted line and the gray box spanning from ±20 dB represents a conservative estimate of the biologically relevant range of dominant ILDs for an adult big brown bat  when the contralateral ear was held at +10 dB (re threshold) and from 47 neurons  when the contralateral ear held at +20 dB (re threshold), and observed three types of ILD response functions across the population of cells tested  and a function that generally dropped to ≤50% of the maximum at both positive  and negative  ILDs. Figure gray box) demonstrating the neuron responded best when sounds were much louder in the contralateral ear.The third and most common type of ILD function we encountered was from cells with monotonic or near-monotonic responses. Cells with monotonic ILD response functions were selective for contralateral sound sources when the function gradually increased from negative to positive ILD values within the biologically relevant range, and were selective for ipsilateral sound sources when the function gradually decreased from negative to positive ILD values within the biologically relevant range. Figure Eptesicus fuscus  ILD response functions at +10 and +20 dB above threshold. The distribution of ILD50 values  ILD functions, we fit a four-parameter sigmoid curve (see Equation 1) to the data from each cell and then measured the ILD value where the 50% response maximum occurred , but at +20 dB re threshold there was a strong trend for a higher proportion of DTNs to show ILD selectivity . We also compared the mean ± standard deviation (SD) electrophysiological response characteristics of DTNs and non-DTNs, but only for those cells with ILD selectivity at either +10 or +20 dB (re threshold), and found no differences in the dorsal-ventral recording electrode depth, BEF, or acoustic threshold. This result indicates that there were no overt topographical differences between DTNs and non-DTNs tested with ILD stimuli in our dataset. There was also no difference in the average duration of the acoustic stimulus that was used to evoke spiking in DTNs and non-DTNs with ILD-selective responses. In Table 50 value for DTNs and non-DTNs with monotonic (or near-monotonic) ILD response functions was located within the biologically relevant range of ILDs for Eptesicus fuscus at both suprathreshold levels. Moreover, there was no significant difference in the mean ILD50 value and slope factor between DTNs and non-DTNs at either level above threshold.Table dotted line and the gray box spanning from ±50 μs represents our estimate of the maximum biological range of ITD cues for an adult big brown bat . Non-selective ITD functions were observed in 24 of 45 cells  tested at +10 dB (re threshold), and 15 of 34 cells  tested at +20 dB (re threshold).The ITD function of a cell shows the number of spikes that were evoked by sounds that were varied in their binaural time of arrival, defined as the onset time of the contralateral stimulus minus the onset time of the ipsilateral stimulus; negative ITD values indicate the ipsilateral signal led the contralateral signal, whereas positive values indicate the contralateral signal led the ipsilateral signal. We recorded ITD response functions from 45 neurons  when the binaural stimulus level was equal in both ears and set to +10 dB (re contra threshold) and from 34 neurons  when the binaural stimulus level was equal in both ears and set to +20 dB (re contra threshold), and observed three types of ITD response functions across the population of cells tested  curves  or +20 dB re threshold . We also compared the mean ± SD electrophysiological response characteristics of DTNs and non-DTNs, but only for those cells with ITD selectivity at either +10 or +20 dB (re threshold), and found no differences in the dorsal-ventral recording electrode depth, BEF, or acoustic threshold. This result indicates that there were no overt topographical differences between DTNs and non-DTNs tested with ITD stimuli in our dataset. At +10 dB (re threshold) there was a trend suggesting that DTNs were tested with shorter duration tones during ITD testing, and at +20 dB (re threshold) this difference was significant revealing that DTNs with ITD selectivity were tested with significantly shorter duration signals than non-DTNs with ITD selectivity. At both levels above threshold there was also a trend for DTNs with ITD selectivity to have higher BEFs than non-DTNs with ITD selectivity  ITD response functions was located outside the biological ITD range for Eptesicus fuscus, whereas this was not true for the population of non-DTNs. Nevertheless, given our small sample size there was no difference in the ITD50 values or slope factors between DTNs and non-DTNs. The fitted four-parameter sigmoid curve for one of the four non-DTNs at +20 dB (re threshold) yielded an extremely large  slope factor that we considered to be an outlier and therefore removed from the population average in Table t-test, p = 0.2030).Table ty Table . It is aSo far our analysis has examined only isolated subsets of cells looking for ILD- or ITD-selective responses at two suprathreshold levels. In this section we explore neural sensitivity to both binaural cues. Within our dataset of 56 neurons we were able to test 43 (21 DTNs) cells for selectivity to both ILD and ITD at +10 dB (re threshold), and 34 (17 DTNs) cells at +20 dB (re threshold). The data in Table 50 and ITD50 values within the biological range for Eptesicus fuscus. A large proportion of neurons with monotonic ILD-selective response functions had ILD50 values within the ±20 dB biological ILD range. At +10 dB above threshold, 16 of 18 DTNs (88.9%) and 13 of 17 non-DTNs (76.5%) had ILD50 values within the biological ILD range. At +20 dB above threshold, 16 of 18 DTNs (88.9%) and 8 of 10 non-DTNs (80.0%) had ILD50 values within the biological ILD range. In our dataset, neurons with monotonic ITD-selective neurons were less likely to have their ITD50 values located within the ±50 μs biological ITD range. At +10 dB above threshold, 1 of 6 DTNs (16.7%), and 2 of 2 non-DTNs (100.0%) had ITD50 values within the biological ITD range. At +20 dB above threshold, 1 of 7 DTNs (14.3%) and 1 of 4 non-DTNs (25.0%) had ITD50 values located within the biological ITD range.We were also interested in determining the number of neurons that had ILDEptesicus fuscus DTNs were as likely as non-DTNs to have binaurally-selective responses. Therefore, in addition to having selectivity to stimulus frequency, amplitude, and duration  of DTNs in normal hearing. Our results show that some DTNs from the IC of the big brown bat have responses that were selective to ILD and ITD binaural cues. Indeed, within the auditory midbrain of Eptesicus fuscus recruited by the separate monaural and binaural central auditory pathways. That study found that the monaural pathways contain all of the circuitry necessary for creating the duration-tuned response physiology  describes how ILD and ITD cues are generated and received by the tympanic membrane as a function of the spatial location of a sound stimulus. And while specific to an individual, the HRTF also varies with sound frequency. For example, low frequency sounds with long wavelengths will diffract more easily around a bat's head and torso, creating small ILD cues at those frequencies. Directional properties of the bat's pinnae and tragus also influence the HRTF through frequency-dependent destructive (attenuation) and constructive (amplification) interference patterns that are generated as the received sound energy reflects off the pinnae and tragus through the external auditory meatus and into the auditory canal .The range of biologically relevant ILDs in Eptesicus fuscus we found that in 29 of 35 cells (82.86%) with monotonic ILD response functions tested at +10 dB (re threshold) the steepest slope of the curve fell within the biological range of ILDs available to the bat  ITD response functions may contribute to the process of sound localization if the steepest slope of their ITD curve passes through the biologically relevant range of ITDs . Similarly, when we varied the stimulus ITD, the ILD was held constant at 0 dB which was also suggestive of a sound source located somewhere along the auditory midline . When animals localize sounds in the real world, ITD and ILD cues co-vary and are concordant with one another along with other localization cues . A concordant combination of all sound localization cues might alter the response properties of directionally-selective neurons in the IC; however, the duplex theory of sound localization (Rayleigh,  Pollak,  and 18 μ Pollak, , respect Pollak, . Therefo Pollak,  rather t Pollak, . AlthougPrior to this report, the responses of auditory DTNs were known to be selective to stimulus frequency, amplitude and, of course, duration. Here we show that some DTNs from the IC of the big brown bat have responses that are also selective to binaural ILD and ITD cues, suggestive of some role in sound localization. Moreover, we found that DTNs were as likely as non-DTNs to have responses that were selective for binaural ILD and ITD cues.50 values and slope factors did not differ between the two populations. We also found that the steepest slope of a neuron's ILD response function fell within the biologically relevant range of ILDs for the big brown bat, suggesting that responses of these cells could serve a role in sound localization.We compared the selectivity of DTNs and non-DTNs with monotonic ILD response functions and found that the distribution of ILDEptesicus fuscus had some form of ITD selectivity. Moreover, DTNs were as likely as non-DTNs to exhibit ITD-selective response functions. It is not clear if the ITD selectivity we observed was simply a by-product of the neural mechanisms underlying ILD selectivity, or if midbrain neurons with ITD-selective response functions are used by echolocating bats to determine the spatial location of sound sources.Although ITD selectivity is thought to be more important in low-frequency hearing, we found that nearly half of the IC neurons we tested in Given that both DTNs and non-DTNs from the IC of the big brown bat had responses that were selective to either ILD, ITD, or both ILD and ITD binaural cues, this suggests that these neurons may function in neural circuits that compute sound source location. In both echolocating and non-echolocating animals, including humans, DTNs could function as level-dependent, spatio-spectro-temporal auditory filters.Manuscript conceived by Riziq Sayegh and Paul A. Faure. Data collected by Riziq Sayegh and Brandon Aubie, and analyzed by Riziq Sayegh and Paul A. Faure. Manuscript written and revised by all authors.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.


The DFT/B3LYP/HuzIII-su3 level of theory was employed. The 13C shielding constants of guest molecules are close to available experimental data. In two cases (the ethane in 512 and the propane in 51262 cages) the 13C shielding constants are reported for the first time. Inversion of the methyl/methylene 13C and 1H shielding constants order is found for propane in the 51262 cage. Topological criteria are used to interpret the changes of values of NMR parameters of water molecules and they establish a connection between single cages and bulk crystal.Calculations of NMR parameters (the absolute shielding constants and the spin-spin coupling constants) for 5The online version of this article (doi:10.1007/s00894-014-2511-2) contains supplementary material, which is available to authorized users. The most intensively studied representatives of clathrate hydrates are those encapsulating methane and carbon dioxide molecules. Abundant in nature, methane clathrate hydrate is recognized as a potential energy resource [2 being one of the most important nowadays [Clathrate hydrates are solid structures composed of a water lattice forming cages in which guest molecules reside. In these structures, the guest molecules . They are made up of 24 polyhedral cages: eight large 16-sided cages 51264 and 16 pentagonal small 12-sided cages 512. We would like to address here the structure I (sI) and structure II (sII) [The crystalline structure of the clathrate hydrates (CHs) is made up of H-bonded water molecules forming a network with cages of different shapes and sizes. The small 5II (sII) , 8 with 4 and CO2 and their mixtures in cages of the hydrate clusters. The change of the frequencies and band shape upon variation of host molecules have been observed. Recently, experimental Raman spectra were published: Ohno et al. [4 of 51262 cages were shifted to lower frequency. Complementary to experimental results density functional theory (DFT) with a dispersion correction have been employed by Ramya et al. [4 and H2O in static-type 512 and larger 51262, explicit cages. Moreover, molecular vibrations were studied by abinitio molecular dynamics simulations with the Car-Parrinello method also by Hiratsuka et al. [12 and larger H structure 435663. The vibrational frequencies for methane are lower in the large cages than those in the small ones, in agreement with experimental Raman data.Spectroscopic methods with quantum chemical calculations provide a valuable tool for the study of hydrates properties from a molecular viewpoint which complement thermodynamic and kinetic studies –13. Moleo et al.  found tha et al.  to charaa et al. , 18 for NMR spectra are a valuable source of information on the molecular and the electronic structure, the conformational changes of a molecule and its environment. Chemical shifts are also widely used to understand the nature of the guest-cage interactions and dynamics of guest molecules in clathrate hydrate for several guest molecules as xenon , 20, met4 and CO2 molecules significantly. Recently, the experimental 13C chemical shift has been used for identification of ethane and propane in the 16-hedral cages of the type sII [13C chemical shifts of methyl and methylene carbons in propane molecule as a guest to gaseous propane at room temperature and atmospheric pressure.We have demonstrated the use of calculated NMR parameters for the analysis of molecular interactions of the methane and carbon dioxide with host-water molecules in static hydrates , 43. As type sII , 45. The12 and larger 51262 cages [1262 cages are filled with ethane, for a long time it was believed that small 512 cages remain empty. However, Udachin et al. showed [13C NMR spectra recorded to date did not show signals attributable to ethane residing in the small cavity, which can be explained in terms of sensitivity of this method. Propane on its own forms a sII hydrate structure, in which bigger 51264 cages are occupied and smaller 512 cages are left empty [Pure methane is known to form sI clathrate hydrate, in which it occupies the vast majority of small 562 cages . Pure et. showed  that a f. showed . Howeverft empty , 49, 50.4, C2H6 and C3H8. It is already known that changing concentrations of CH4, C2H6 and C3H8 and pressure-temperature conditions, both sI and sII structures could be obtained [1262 and 51264 cages.As natural hydrates are almost always formed by mixtures of lower hydrocarbons and other molecules, many studies were already devoted to analysis of hydrates formed by binary and ternary mixtures of CHobtained –53. Thos12 cage of sI or sII structure or propane in 51262 cage of sI structure. Moreover, neither 1H shielding constants for all guest molecules of aforementioned hydrates nor spin-spin coupling constants for them could be found in the literature. Moreover, the NMR spectra were not measured due to limitations of available experimental methodology. It is expected that with the advent of more sensitive NMR experimental set-ups and methods the calculated results undertaken by us will be verified experimentally.Those results were obtained from thermochemical or X-ray studies. The authors did not find any NMR data for ethane residing in 52 clathrates [This work is a continuation of our previous study of water clusters , methaneathrates . The cagathrates ; (ii) thathrates , 17, 19)athrates , 56 (as athrates ); (iv) iathrates . Our aimThe structure of this paper is as follows: the method section employed for geometry optimization and the calculations of NMR parameters are described in Section 12 (found in sI and sII hydrate structures), 51262 (of sI clathrate hydrate) and 51264 (of sII clathrate hydrate). The locations of the water’s oxygen atoms residing in the vertices of the cages were based on X-ray data [As a model systems we have chosen cages 5ray data , 56. As ray data  and the 2 hydrates [We have applied a geometry optimization scheme previously used for the COhydrates  and simihydrates . Thus:  and 1 % solution of TMS in CDCl3 (183.94 ppm) by Jackowski and Makulski [1H shielding constants are referenced to benchmark values for gaseous molecules by Garbacz et al. [4, C2H6 and C3H8 guest molecules and hydrogen-bonded water network forming cages are predominantly of dispersive character, the main factors affecting shielding constants are the size and shape of the cage. The size of the cages determines, whether guest molecules could rotate inside. Symmetry of the cavity also plays a part as it may favour distinct orientations of guest molecule in cages big enough to provide rotational freedom to the guest molecule. Different orientations of the guest could result in different shielding constants of nuclei forming quest molecule.The Makulski . The 1H z et al. , obtaine4, C2H6 and C3H8 are recorded for 13C nuclei .Most experimental data  for clathrate hydrates containing CH13C shielding constant is growing with the cage size toward the value of gaseous guest molecule . The calculated values are -6.02 ppm, -4.75 ppm and -3.26 ppm [13C chemical shielding connected with the enclathration are systematically greater than experimental ones by more than 2 ppm. Much better quantitative agreement is found for the changes of the 13C shielding constant between cages. Going from the cage 51264 to the cage 51262 and further the cage 512, the 13C shielding constant is changing by -1.50 ppm and -2.40 ppm  and -1.49 ppm and -1.27 ppm .Let us first discuss shielding constants . The differences between cages are -1.70 ppm (experiment) and -1.12 ppm  . Again, as was already seen for the methane, computed changes of the 13C shielding constant between cages are quantitatively accurate. Moreover, they are very close  for both ethane and methane molecules enclathrated in 512, 51262 and 51264 cages.Analogous trends are found in this paper for the ethane molecule. The experimental enclathration changes of the 13C shielding constants obtained for the methane and the ethane show that the size of the cage is the main parameter influencing shielding constant of their carbon atoms. Symmetry of the cage seems to be less important, what is understandable in a connection to the symmetry of the quest molecules. Computed values of the shielding constants’ anisotropies confirm this finding. Thus, the anisotropy of the shielding constant of the carbon nucleus in the cages would also be of interest. Tetrahedral symmetry of the methane molecule together with roughly spherical environment inside 512 and 51264 cages do not favour any particular orientation of guest molecules. Although the 51262 cage provides an oblate cavity, its distortion from sphericity is too small to affect the shielding constant of methane carbon. Values of the static anisotropy  confirm this statement. The experimental anisotropy is expected to be equal zero. On the other hand, the axial symmetry of the ethane results in its orientation in oblate 51262 cage parallel to hexagonal faces. However, the static anisotropy calculated for the ethane molecule in the 51262 cage (6.88 ppm) is much smaller than that found for the CO2  for the 51262 cage.The 2.96 ppm ), which 12 cage, therefore we have calculated the NMR properties for bigger cages 51262 and 51264  - too l3H8 and compare them with these values for the methane and the ethane. The shielding constant values for13C for methyl group form growing trend for both experimental and calculated values . The enclathration in the 51264 lowers absolute shielding constant by 1.6 ppm according to experimental data [1264 and 5.9 ppm for 51262. It is analogous to what was previously described for methane and ethane molecules in terms of trend as well as relative changes of the shielding constant values.Let us discuss now the shielding constants calculated for methyl group of the Ctal data , while c13C NMR spectrum showed two peaks corresponding to methyl and methylene carbon nuclei. In this paragraph we would like to discuss the changes of methyl and methylene shielding constants attributed to the studied cages: 1264)) by Kida et al. [1264 cage  and in the opposite direction than shielding constant of methylene carbon (0.9 ppm) and is able to reverse the sequence of their values . That is the cause why reversed sequence of shielding constant is not reproduced for the 51264 cage.Looking for the source of this divergence, let us analyse now changes upon the complexation, i.e. the transfer from the gas phase to clathrate. Experimental 1H absolute shielding constants calculated for gaseous guest molecules are all greater by 1 ppm than the corresponding experimental results. These calculated values will be now discussed , but correlation with trend for methylene carbon shielding constants supports correctness of this ordering.The12 and 51262 enclathrating methane are taken from our former publication on the NMR of sI methane hydrate [Shielding constants for oxygen and hydrogen atoms of water molecules forming cages are shown in Table  hydrate , in whic17O shielding constant with growth of the size of a molecule inside the cavity? The answer one can get looking on the data in Table 17O shielding constant are getting lower for both DAA and DDA molecules Let us start analysis of the data in Table 17O shielding constant between the DDA and DAA types of water molecules. The changes between 51262 and 51264 for DAA type water molecules are equal to 2.76 ppm and 2.96 ppm for cages containing the ethane and the propane, respectively. Analogous changes for DDA type water molecules are equal to 5.97 ppm and 6.62 ppm. Accordingly, the changes for cages enclathrating both guests are quite close and DDA water molecules are more affected by the growth of the size of the cage. Moreover, similar values of 17O shielding constants for DAA and DDA groups of water molecules observed for cages independent of the type of guest molecule results also in similar differences between 17O shielding values of DAA and DDA type. For 51262 cage those differences are 2.03 ppm, 1.20 ppm and 1.36 ppm for the methane, the ethane and the propane, respectively, while analogous changes for cage 51264 are 5.16 ppm, 4.41 ppm and 5.02 ppm.Secondly, the question may be asked, should there be any change of the17O shielding constants and the strength of H-bonds in clathrate hydrates. The observed monotonic growth of the 17O shielding constants may be correlated with decreasing strength of average interaction between water molecules for growing cages. It is analogous to what was previously seen for CO2 clathrate cages [17O shielding constants based on H-bond characteristics presented above.Thirdly, we considered a correlation between the te cages . It was 2 clathrate hydrates [6,5561 and 5462. In single cages those three dimensional topologies are reduced to 53 and 5261 topologies, what was described in detail in [12 cage all water molecules are of 53 topology, while for the 51262 and 51264 cages both 53 and 5261 topologies are present. As to 5561 molecules, half of them is attaining 53 topology, while second half - 5261. This division comes from the fact, that in the crystalline structure the cages of 512 type are neighbouring those of the 51262 type. Therefore, some water molecules forming the pentamers of 512 cages are at the same time forming the hexamers of 51262. However, taking single 512 cage, this information is lost - they are all of the 53 topology. Similar point is true also for the 51262 cage - in CHs some of its water molecules forming only pentamers are located in a junction with neighbouring 51262 cage and form its hexamers, so the 53 topology of single cage is represented by the 5561 in crystal. For sII structure situation is simpler. All 56 of crystal are attaining 53 topology. As for 5561, half of them is reduced to 53 and half to 5261 topologies, analogous as for sI structure. Therefore there is no direct relationship between topologies found in crystal and single cages for any of sI and sII structures. Those differences in H-bond framework and topology between single cages and three dimensional structure should be considered to properly compare our results with experimental ones.To continue the answer on above question, much more straightforward for this purpose is analysis based on topological criteria (see our former paper on COhydrates ). Crystaetail in . For the17O shielding constant are growing with the cage size for both 53 and 5261 water molecules. The changes between 51262 and 51264 for 53 water molecules are equal to 3.02 ppm and 3.40 ppm for cages containing the ethane and the propane, respectively. Analogous changes for 5261 water molecules are equal to 4.45 ppm and 4.86 ppm. The change in oxygen shielding constants for 53 water molecules going from 512 to 51262 cage enclathrating the methane is 0.35 ppm. It is not possible to obtain such changes for 5261 as all water molecules forming 512 cage are of 53 topology. Generally, a growing trend observed for the 17O shielding constants for both types of topologically distinguished water molecules is analogous in the direction and scale observed in H-bond perspective.To continue the analysis, the17O shielding constants should be expected. In some cases they amount to several ppms, so with increasing sensitivity of the NMR instrumentation and an efficiency of data acquisition they should be observable experimentally in the future.The trends described above were observed for single cage, so the question arises - how do they correspond to the three-dimensional lattice of crystalline hydrate? As crystals of hydrates contain water molecules of distinct topologies, analogous to those found in single cages, similar differences betweenOxygen atoms were divided into two categories in the H-bond network perspective, namely those belonging to DAA and DDA water molecules. For hydrogen atoms three categories are needed, as for DAA water molecules one of the hydrogen is involved in H-bond formation and second is not (dangling hydrogen). The values of shielding constants are presented in Table 17O shielding constants. That is: (i) the σ(1H) is increasing with the growing size of the cage, (ii) the σ(1H) is almost unaffected by the enclathrated molecule for all cages, (iii) the differences between distinct types of hydrogen atoms are comparable for all cages. The observations made for 17O shielding constants in topological perspective also hold for 1H values, but respective changes are much smaller and trends less clear.Generally, for all three categories of protons conclusions are analogous to those drawn for respective12 cage bond lengths are affected only slightly (thousandths of Å). Angles between bonds are more affected, but the overall changes of the SSCC caused by enclathration are small. All 1J couplings are dominated by the FC term and variations in its values are mirrored in variations of the SSCC. The PSO and DSO values may make up to 20 % of total SSCC, but as they are opposing in signs they mostly cancel each other out. Changes of their values connected with the enclathration may be appreciable, but again - in opposite directions, so the influence on total SSCC value remain limited. The SD term values are small and almost unaffected by enclathration. Electronic Supplementary Information contain tables summarizing values of SSCC, four distinct terms and interatomic distances.The changes of the SSCC of guest molecules induced by enclathration are caused by distortions of geometry and the interactions with host water molecules. Even for the smallest 52 hydrates [1262 cage enclathrating ethane molecule. This choice seem to be most representative for all cases, as ethane could be enclathrated in all cages and 51262 cage could contain all three guest molecules described in this paper.Inter- and intramolecular spin-spin coupling constants for water molecules forming cages may be correlated with strength of interactions those molecules are involved in. For non-polar guest molecules interaction with quest is rather small and dominated by interactions between water molecules, among which H-bonds are by far the strongest. Therefore, it is possible to correlate strength of H-bonds between water molecules forming cages with SSCC characterising those water molecules, as was already done for methane and COhydrates , 43. PatAs was already mentioned, DAA-DDA scheme does not provide straightforward connection between single cages and three-dimensional crystal structure. Topological criteria are more reliable for this purpose. However, patterns observed for water SSCC in the H-bond perspective are more explicit than those revealed by topological one. Therefore we will not discuss them in the main body of the article - all data could be found in respective tables in Electronic Supplementary Information.1JOH and 2JHH coupling constants and their components are presented in Figs. 1JOH couplings are dominated by the FC term, while among others only PSO gives a non-zero contribution. A slight decrease of the absolute value of the shielding is noted for all water H-bond patterns.Intramolecular 2JHH coupling constants are also dominated by the FC term. As opposed to 1JOH, also PSO, DSO and SD contributions are non-negligible, but as the sum of their values is close to zero, final coupling is again close to the FC value. For both DAA and DDA water molecules 2JHH coupling constants are lowering with the interatomic H ⋯H distance  (more data in Electronic Supplementary Information).Intramolecular h2JOO coupling constants and their components are presented in Fig. Intermolecular h1JOH coupling constants for all four possible pairs of water molecules forming H-bonds, together with their components are presented in the last Fig. h1JOH coupling constants are also different. In this sense it may be stated, that h1JOH coupling of (O)DDA-(H)DAA type is weakest, while h1JOH coupling for three other H-bond types are of comparable strength. This relation was already observed for carbon dioxide clathrate hydrate [h1JOH and average O ⋯H distances for distinct H-bond types are very close to previously noted, what augments credibility of those conclusions.Intermolecular hydrate . Moreove12, 51262 and 51264 of sI and sII clathrate hydrates containing the methane, the ethane and the propane as guest molecules, were performed at DFT/B3LYP/HuzIII-su3 level. Influence of enclathration on guest molecules NMR characteristics was discussed. Two perspectives of the interpretation of the shielding constants and the SSCC of water molecules, based on H-bond characteristics (DDA and DAA water molecules) and topological criteria, were presented. The connection between the calculated and experimental results was described. The most important findings of this paper are:13C shielding constants for the methane, the ethane and the propane inside cages studied are determined. In two cases (ethane in 512 and propane in 51262 cages) neither experimental nor theoretical data existed. Shielding constant values for 13C for methyl group form growing trend for both experimental and calculated values .The absolute1262 cage. It should be verified experimentally in the near future.The change of the methyl/methylene order of the absolute shielding constants is found for the propane enclathrated in 51H shielding constants of the guest molecules for all studied cages are presented for the first time. Analogously to 13C shielding constants of propane in 51262 cage, change of the methyl/methylene order of the absolute shielding constants is found also for the 1H nuclei.The absolute 17O shielding constants for the DAA and DDA groups of water molecules observed for cages independent of the type of the guest molecule results also in similar differences between 17O shielding values of the DAA and DDA type.Similar values of the 17O absolute shielding constants may amount to 2 ppm in single cages. It may be expected, that similar differences could be found in real crystals.The division of water molecules forming host lattice according to topological criteria enables most direct connection between single cages and bulk crystal. Differences of the 12 cage and the propane in 51262 cage) our results are first in the literature presenting 13C shielding constants of guest molecules. Additionally, all 1H shielding constants and all intramolecular spin-spin coupling constants for the guest molecules were not published yet. Therefore these results should be helpful in an interpretation of future experimental data.Until now no experimental data exists for some of the structures discussed in this paper, as respective structures were simply not synthesized yet or their spectra could not be obtained to date due to limitations of available experimental NMR techniques. In those cases  for all molecules forming cages 5(PDF 229 KB)Below is the link to the electronic supplementary material.