Troglodytes aedon, at high and low elevations in California and Costa Rica, collecting data on clutch size, the duration of incubation and nestling periods, parental attentiveness, nestling growth rate, and nesting success. Our data support results from cross‐species comparisons, but also revealed unanticipated results from low elevation temperate zone house wrens in the southwest. This population had prolonged incubation and nestling periods similar to those found in the tropics. We also found that temperate zone females, especially those at our higher elevation site, spent more of their day incubating than did tropical females. Nest temperature at our high elevation temperate zone site was higher than that at all other tropical sites. Age at fledging did not differ between sites. Total feeding rates per chick and male feedings per chick did not vary between sites. Nest success rates showed the predicted effect of latitude, but not the predicted effects of elevation. Our results extend low elevation house wren research into the southwestern US and contribute the first intraspecific elevational comparison in the Neotropics. Data from our low elevation southwestern site present a unique suite of life history traits that align more with tropical house wrens, although with a larger clutch size, and point to food limitation and/or high predation pressure as being possible drivers of some of these differences. These results highlight the need for additional studies of house wrens and other broadly distributed species at a more diverse array of sites to better understand which forces drive the evolution of different life history strategies across major biogeographical gradients.While cross‐species comparisons of birds suggest that as latitude decreases or elevation increases, clutch size decreases and the duration of developmental stages and parental attentiveness increases, studies comparing populations of the same species are rare. We studied populations of house wrens,  Troglodytes aedon), at high and low elevations at temperate and tropical latitudes. It extends extant research on this species into the southwestern US and provides the first comprehensive data with which to compare reproductive biology at different elevations in the Neotropics. Results from the low‐elevation southwest population differ from results of studies conducted elsewhere in the US and align more with tropical wrens. These results call for more diversity in the selection of study populations and provide new insight into the selective pressures underlying life history evolution.This study presents the first comparison of the reproductive biology of a single species, the house wren ( Life history theory provides a framework to explore this balance by generating a set of rules that suggest how organisms have evolved to optimally allocate their resources in different environments Roff, . Yet, deResearch on birds has played a significant role in the development of life history theory Lack, . ObservaEffects of elevation on life history traits have received far less attention than effects of latitude, and, further, variation in elevation effects between different latitudes has not been directly examined. Nonetheless, several cross‐species comparisons Badyaev,  and studTroglodytes aedon, offers a relatively unique opportunity for comparative work as it is distributed throughout North, Central, and South America at a variety of elevations,although its status as a single species has been called into question . Here, house wrens were primarily associated with small willow‐ and aspen‐lined streams. Our low‐elevation site was located 396 km south in Marshall Canyon (“temp‐low”) in La Verne, CA . Here, we conducted observations throughout the entire breeding season, from late March through mid‐July, 2003 to 2008. This site experiences a Mediterranean climate and consists of riparian oak woodlands and coastal sage scrub. Marshall Canyon is a refuge for house wrens and other birds, as well as large and small mammals, as it is one of the few canyons in the region with a year‐round small stream. While our high and low sites differ slightly in latitude, their difference is below the 5° threshold used by Boyle et al.  at two locations in California. In both the southern California lowlands and the Eastern Sierras, we found house wrens to be distributed in relatively small populations, associated with lakes and streams, presumably due to drier, warmer conditions. We studied a high‐elevation population from May to early July 1997–2003 and 2005 at the Sierra Nevada Aquatic Research Laboratory , 13 km east of Mammoth Lakes, CA, from 1997 to 2003, and in 2005  in 2001 and 2004. Our low‐elevation site was located 87 km east in cattle ranches at the edge of lowland tropical rainforest near La Selva Biological Station in Puerto Viejo de Sarapiquí . Here, observations were conducted in 2002 and 2004. At both sites, some birds were breeding before we arrived in mid‐January of each year, and some were still breeding when we left in mid‐July; our field seasons covered the breeding peak at both sites.Our tropical sites were located in Costa Rica. In the tropics, house wrens are likely to be found in disturbed habitats and have been studied at two lowland sites in Panama—cattle ranches Freed, , and a nDuring the first year at each site, nest boxes were attached to trees at each location where a house wren was singing. Boxes were replaced as needed in the second observation year. The second year at each tropical site (2004) was used for cross‐site comparisons, since boxes were in place before breeding began. The same year was selected for these comparisons for our temp‐low site, except for egg weights as noted below. Data were not collected in 2004 from our temp‐high site, so data from 2003 were used in cross‐site comparisons.No antipredator devices were attached to the boxes. Nest boxes at all sites were checked for evidence of reproductive activity every other day. As predicted dates of hatching and fledging approached, we began checking boxes daily to determine exact dates of hatching and fledging. Incubation duration was calculated using the day of clutch completion as Day 0 through the day the first chick hatched. The duration of the nestling period was calculated with the day of first hatch as Day 0 through the day in which chicks fledged.As early as possible in the season, adults on each site were mist‐netted, given a unique combination of three colored plastic bands and one numbered aluminum band, weighed, and wing cord and bill measurements taken. Netting was avoided during egg laying; some adults were weighed during nestling feeding if they had not been caught previously. All eggs in a nest were weighed the day after clutch completion. All nestlings were weighed on Days 5 and 10 post‐hatch when possible, and each nestling was given a numbered aluminum band before fledging.A nesting attempt was considered successful if at least one nestling fledged. In all cases where clutches completely disappeared, evidence suggested either predation by snakes  or mammals (nest destroyed/structure disrupted), but this could not be directly confirmed.2.2Onset HOBO Pro Series data loggers were attached to a subset of nest boxes at each site before or during the egg‐laying period to monitor female behavior. Ambient temperature probes from each logger were screwed onto the outside bottom of the nest box, and the internal probe was woven through the nest lining until it was at the nest cup surface within 0.5 cm of the eggs. Internal probes were set to maximize detection of arrival and departure of the female from the nest, and are thus likely to provide a relative, but not absolute measure, of nest temperatures at each site. Loggers were programmed to monitor both ambient and internal nest temperature at 30‐second intervals throughout the duration of incubation. The beginning or end of an incubation bout was recorded when the internal temperature probe changed more than 0.5°C within a 30‐s period . For each feeding measure, we calculated “early” and “late” feeding averages in the nestling period by using the mean of observations on Days 3 and 5 (Week 1) and Days 11 and 14 (Week 2), respectively. This analysis only included pairs for which repeated observations were available in the first and second week postfledging.Each author collected data at both temperate and tropical sites; three of the authors collected data and/or trained and supervised data collection and behavioral observations at all four sites.Protocols were approved by the Institutional Animal Care Committees of Pomona College and the University of California Santa Barbara. Research in Costa Rica was conducted under permits from the Costa Rican Ministry of the Environment and Energy (MINAE) and overseen by the Organization for Tropical Studies.2.4t‐tests. A coefficient of variation ([mean/SD] × 100) was also calculated to estimate interyear variability in each parameter at each site. For comparisons between sites, we used the year in which both tropical sites were observed, 2004, and the closest year for which comparable data were available for the temperate sites . The one exception was a comparison of nesting success rates where we used all nest data from all years to permit statistical analysis. In this analysis, nests were considered successful if at least one nestling fledged from a completed clutch. This measure of “apparent success” was possible because we were able to monitor all the nests of singing males at each site; this measure is used in comparisons across house wren studies , simple slope analyses using pooled variances were used for follow‐up pairwise comparisons.To avoid any possible effect of seasonal progression on breeding behavior, only first clutches for which data were available were used in analyses involving clutch size, incubation duration, feeding rates, fledging age, and weights of eggs and chicks. We used mean weights per clutch when comparing chick and egg weight between sites. All data were tested for skewedness and kurtosis before analysis and found to be normally distributed. For behavioral, reproductive, or morphological parameters, we conducted comparisons between sites using a two‐way analysis of variance, with latitude and elevation as fixed factors. A repeated measures two‐way ANOVA was used to analyze feeding behavior across time. If a two‐way ANOVA was significant , although the interyear variability in clutch size for our temp‐low site was lower than that for other sites , whereas both incubation and nestling periods differed between years at both temperate zone sites . Coefficients of variation for duration of incubation and the time to fledge indicated higher variability in temperate than tropical bids  and low elevations (p < .001), only tropical birds showed a significant effect of elevation on clutch size , such that high‐elevation birds had smaller clutches than low‐elevation birds.A comparison of breeding parameters across sites within a single year showed varied effects of latitude and elevation Figure , Table 2p < .001, mean diff = 2.57 days), but there was no effect of latitude when comparing low‐elevation sites (p = .822). In addition, also as a result of the long incubation duration at our temp‐low site, we found that incubation duration decreased with elevation in the temperate zone (p < .001), whereas in the tropics, incubation duration increased with elevation (p < .001).We found main effects of both latitude and elevation on the duration of incubation Figure  and 3. Hp = .006), but not low‐elevation birds (p = .300). Within a given latitude, there was an effect of elevation on age at fledging in tropical birds (p = .015), but not in temperate zone birds (p = .161).We found no main effects of latitude or elevation on age at fledging Figure  and 3, b3.3F = 16.501, df = 1, p = .002). There was no effect of elevation on success rate , and there was no significant interaction between latitude and elevation . Nest success at high‐elevation sites appeared to be more variable than it was at low‐elevation sites . All nests that failed after clutch completion appeared to fail due to predation at all sites. There was no evidence of death by starvation or injury; rather, evidence suggested that disappearance of eggs or chicks was due to predation by mammals (highly disturbed or destroyed nest) or snakes (undisturbed nest). When looking across years, we found a main effect of latitude on nesting success, with lower nest success in tropical birds than temperate birds  successfully produced two clutches. In the tropics, at least 18% of high‐elevation pairs and 15.6% of low‐elevation pairs successfully produced more than one clutch during the period in which they were observed.3.4F = 49.18, df = 1, p < .001) and elevation , such that females were active longer at higher latitudes and elevations . There was no interaction between latitude and elevation . Analysis of the average percent of the day that females spent in the nest box also showed main effects of both latitude and elevation (Tables p < .001), whereas tropical females showed no effect of elevation (p = .258). In addition, an effect of latitude on time spent incubating was only evident when comparing birds at the two high‐elevation sites (p < .001); the two low‐elevation sites did not differ in how much of the day was spent incubating (p = .268).To examine daytime incubation behavior, we considered the activity of females between their first departure from nest box and their last return. The amount of time between when females first left and last entered the nest, a measure of active day length, showed main effects of latitude  and elevation  and no interaction effect . These effects were due primarily to the large impact of females on the nest at our temp‐high site .Incubation behavior is best understood within the context of ambient temperature at each site Table . At nighp = .019), but not in the tropics (p = .163), and high‐elevation nest temperatures differed between latitudes (p = .01), whereas low nests did not (p = .170). The difference between daytime average box and ambient temperature showed a main effect of latitude , with a larger difference between ambient and box temperatures in the temperate zone, as well as a main effect of elevation , with larger differences at higher elevations. There was no interaction effect . Again, this was the result of the large impact of female presence in the nest box at our temp‐high site .During the daytime, ambient temperature was higher at lower latitude and lower elevation sites Table . Daytime3.5We began our investigation of chick development by considering the relative size of adults at our different sites Table . SignifiHouse wren eggs showed a main effect of latitude Tables  and 5 anp = .594) whereas all other comparisons were significant (p < .001). Growth rate, measured by percent increase in weight between 5‐ and 10‐day‐old nestlings, did not show an effect of latitude , whereas low elevations did not (p = .065). Both temperate (p = .008) and tropical wrens (p = .045) show an increase in total number of feedings with an increase in elevation.Feeding trips to the nest made by males and females were compared between early (Week 1) and late (Week 2) in the nestling period Table . We usedp = .038) and temp‐high birds (p = .046). All other comparisons between nestling stage, latitude, and elevation were not significant at p > .05.When the number of feeding trips was adjusted for the clutch size (“feeds per chick”), we again found a main effect of nestling age on parental feeding behavior Tables  and 7, bp = .012), as well as less than that for temp‐high males (p = .027). A nestling age × latitude × elevation interaction was not significant , but a comparison of low‐elevation males at different latitudes was not significant (p = .917), and temp‐low males fed less than temp‐high males (p = .004), while trop‐low males fed more than trop‐high males (p = .048). There was not a significant nestling age × latitude × elevation effect  or higher food quality.4.3Our results on tropical house wrens confirm a suite of traits that characterize a strategy that includes a longer breeding season with multiple breeding opportunities per pair, bigger adults with shorter wings, smaller and fairly stable clutch size, larger eggs and nestlings, longer periods of incubation and fledging, less time per day spent in incubation, and fewer feeding visits per nest. Our ability to compare tropical wrens directly with our temperate populations was complicated by the fact that our low‐elevation temperate zone site differs from other sites in the US in which house wrens have been studied . However, latitudinal comparisons between high‐elevation sites yielded results consistent with expectations.House wrens at our tropical sites also provided an opportunity to compile a broad array of traits that vary with elevation, adding the Neotropics to the small list of regions in which elevation effects have been studied  and tropical populations (T. a. musculus) is essential.The temp‐low site, Marshall Canyon, yielded provocative results. Marshall Canyon house wrens had large clutches, smaller adults with longer wings, and smaller eggs and young, like most temperate zone birds that have been studied. However, Marshall Canyon wrens also had longer incubation and nestling periods, less time on the nest, lower feeding rates, and low nest success rates, which are more typical of tropical birds. It is unclear whether these differences are due entirely to local conditions, a different evolutionary history and taxonomic status than house wrens further east, or both. Of the selective pressures proposed to drive different strategies, food limitation would seem a likely explanation of the lower rates of female incubation and parental food delivery and at least one unpublished food supplementation study  suggests that this may be the case. A high predation rate, leading to a strategy of fewer visits to the nest to incubate and feed young, is another possible explanation. Testing between selective pressures at this and other sites in this region and comparisons with other Mexican ; data curation ; formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); resources (lead); software (lead); supervision ; validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing . Stephanie M. Correa: Data curation ; investigation ; methodology (supporting); project administration ; resources (supporting); software (supporting); supervision ; validation (supporting); visualization (supporting); writing – original draft (supporting); writing – review and editing . Kate A. Freund: Conceptualization (supporting); data curation ; formal analysis ; funding acquisition (supporting); investigation ; methodology (supporting); project administration ; resources (supporting); software (supporting); supervision ; validation (supporting); visualization (supporting); writing – original draft (supporting); writing – review and editing (supporting). Matthew J. Fuxjager: Conceptualization (supporting); data curation (supporting); formal analysis (supporting); funding acquisition (supporting); investigation ; methodology (supporting); project administration ; resources (supporting); software (supporting); supervision ; validation (supporting); visualization (supporting); writing – original draft (supporting); writing – review and editing .Student participation on this project was supported by the Pomona College Summer Research Program and the NSF Research Experiences for Undergraduates Program.The authors have no conflicts of interest. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the U.S. Fish and Wildlife Service.


Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The enzyme controls whether cells utilize PA to produce membrane phospholipids or the major storage lipid triacylglycerol. PA levels, which are regulated by the enzyme reaction, also control the expression of UASINO-containing phospholipid synthesis genes via the Henry (Opi1/Ino2-Ino4) regulatory circuit. Pah1 function is largely controlled by its cellular location, which is mediated by phosphorylation and dephosphorylation. Multiple phosphorylations sequester Pah1 in the cytosol and protect it from 20S proteasome-mediated degradation. The endoplasmic reticulum-associated Nem1-Spo7 phosphatase complex recruits and dephosphorylates Pah1 allowing the enzyme to associate with and dephosphorylate its membrane-bound substrate PA. Pah1 contains domains/regions that include the N-LIP and haloacid dehalogenase-like catalytic domains, N-terminal amphipathic helix for membrane binding, C-terminal acidic tail for Nem1-Spo7 interaction, and a conserved tryptophan within the WRDPLVDID domain required for enzyme function. Through bioinformatics, molecular genetics, and biochemical approaches, we identified a novel RP (regulation of phosphorylation) domain that regulates the phosphorylation state of Pah1. We showed that the ΔRP mutation results in a 57% reduction in the endogenous phosphorylation of the enzyme , an increase in membrane association and PA phosphatase activity, but reduced cellular abundance. This work not only identifies a novel regulatory domain within Pah1 but emphasizes the importance of the phosphorylation-based regulation of Pah1 abundance, location, and function in yeast lipid synthesis.The  Saccharomyces cerevisiae PAH1-encoded phosphatidic acid (PA) phosphatase (PAP) is one of the most highly regulated enzymes in lipid metabolism (2+-dependent dephosphorylation of PA to produce diacylglycerol (DAG) (via the liponucleotide intermediate CDP-DAG or the major storage lipid triacylglycerol (TAG) via the intermediate DAG  regulatory circuit  , 5  . The enziate DAG . The expal phase  when celal phase , 8. As cal phase  as energal phase , 8. PA l circuit , 11, 12.via the CDP-DAG pathway, whereas mutants defective in this pathway  and, in general, the phosphorylated enzyme is localized to the cytosol , while others  are common to multiple protein kinases . Some phosphorylations are hierarchical in nature where the phosphorylation at one site affects the phosphorylation at another site  or inhi85-Pho80 , Rim11 -Spo7 (regulatory subunit) protein phosphatase complex  Fig. 1)Fig. 1). A). The N-LIP and the haloacid dehalogenase (HAD)-like domains are required for PAP activity (XDX(T/V) catalytic motif that is essential to PAP activity and Pah1 function . The interaction between Pah1 and the Nem1-Spo7 complex is mediated by its C-terminal acidic tail (in vivo function of Pah1 but is not required for its PAP activity per se (A). Also of note is that the unphosphorylated form of Pah1 is highly susceptible to 20S proteasome-mediated degradation, but its phosphorylation by multiple protein kinases at sites within the IDRs stabilizes the enzyme against the proteasomal degradation  throughout the primary structure of Pah1 that are important to its location, function, and regulation A. The N-activity , 38. Thetic core . This iniae Pah1 C. The indic tail , whereasdic tail . Within dic tail  that is y per se , 64. Mosy per se  Fig. 2AAA. The Nradation .B). Additionally, the AlphaFold model of Pah1 predicts structure for this region and it is well conserved among multiple fungal Pah1 orthologs. Taken together, these analyses led to the hypothesis that this region represents a functional domain that we named RP (for regulation of phosphorylation). We showed that the ΔRP mutation in Pah1 results in a 57% reduction in the endogenous phosphorylation of the enzyme, an increase in membrane association and PA phosphatase activity, and a gain of Pah1 function in the absence of the Nem1-Spo7 complex. This work not only identifies a novel regulatory domain within Pah1 but emphasizes the importance of the phosphorylation-based regulation of Pah1 abundance, location, and function in yeast lipid synthesis.The stretch between the N-LIP and HAD-like domains of Pah1 has been thought to be a continuous IDR , 65. HowFig. 2S. cerevisiae Pah1 contain IDRs (A) (B). Consistent with previous findings (B). These algorithms also predict a significant stretch  within the middle region of the N-terminal IDR that is not disordered (B). We refer to this region as the RP (regulation of phosphorylation) domain. The position of the RP domain is highlighted in the AlphaFold (C). A BLAST (Large stretches of ain IDRs A 56) pr prS. cerIDRs (A) , flDPnn IDRs (A) , and FolIDRs (A)  to analyIDRs (A) B. Consisfindings , 64 . The ΔRP mutation in Pah1 did not have a major effect on the TAG content of the Nem1-Spo7 complex-containing cells in the exponential phase, but caused a decrease in TAG content in the stationary phase suggesting some dysregulation (A). As described previously . The TAG content in the exponential and stationary phases, respectively, of the cells with WT Pah1 that lack the Nem1-Spo7 complex were 1.6-fold and sevenfold lower when compared with the cells containing the Nem1-Spo7 complex. This highlights the importance of the Nem1-Spo7 complex in the recruitment, dephosphorylation, and activation of Pah1 at the nuclear/ER membrane .The synthesis of TAG is largely dependent on the Pah1 function , and acceviously , 6, the eviously A. The ΔRgulation A. As desf growth B. The TAmembrane . Strikin WT Pah1 B.Figure A). The ΔRP mutation did not affect lipid droplet formation in cells with the Nem1-Spo7 complex. Consistent with the negative effect the nem1Δ mutation has on TAG content, the lipid droplet formation in the stationary phase was compromised by loss of Nem1-Spo7 complex function . However, in cells lacking the Nem1-Spo7 complex, the ΔRP mutation had a positive effect on lipid droplet formation. In both exponential and stationary phase cells the ΔRP mutation in Pah1 resulted in a 1.5-fold increase in lipid droplet formation .We next examined the effect of the ΔRP mutation on the formation of cytoplasmic lipid droplets. As described previously , 72 for Fig. 5S. cerevisiae cells defective in any component of the Pah1/Nem1-Spo7 phosphatase axis exhibit an irregularly shaped nucleus due to the aberrant expansion of the nuclear/ER membrane (vector control). This phenotype is attributed to loss of Pah1 PAP activity, accumulation of PA, and increased synthesis of membrane phospholipids (A). However, as described previously (B). In marked contrast, almost 80% of cells lacking the Nem1-Spo7 complex, but expressing the ΔRP mutant form of Pah1, showed normal round nuclei (B).membrane , 57, 72 holipids , 32, 38.holipids . We obseholipids A. Howeveeviously B. In mard nuclei B.Figure pah1Δ) and without (pah1Δ nem1Δ) the Nem1-Spo7 complex. Cultures were grown to the mid-exponential phase, cell extracts (E) were prepared and fractionated into the cytosolic (C) and membrane (M) fractions, and Pah1 was analyzed by immunoblotting with anti-Pah1 antibody (A). Exponential phase cells were used for this experiment because Pah1 is subject to proteasomal degradation in the stationary phase and the protein is difficult to visualize (A). As expected, in both genetic backgrounds, nearly all WT Pah1 was associated with the cytosolic fraction (B). Furthermore, only about 1 to 2% of the WT enzyme was associated with the membrane fraction (C). These observations confirm the importance of phosphorylation to the localization and abundance of WT Pah1 (B). The lower abundance of the ΔRP mutant protein is not expected to be due to a defect in gene expression since all genetic constructs were expressed from the same plasmid driven by the native PAH1 promoter, but instead due to increased sensitivity to 20S proteasomal degradation. The ΔRP mutation also had a major effect on the cellular distribution of Pah1 (B). The relative amounts of the ΔRP mutant protein associated with the membrane for cells with and without Nem1-Spo7 were 37% and 28%, respectively (C).The effects of the ΔRP mutation on the abundance and cellular location of Pah1 were examined. The WT and ΔRP mutant forms of the enzyme were expressed from the single-copy plasmid in the cells with . Preserving the hyperphosphorylated state of the enzymes was ensured by expressing them in cells  lacking the Nem1-Spo7 protein phosphatase complex . The ΔRP mutation did not have a significant effect on the kinetic properties of the unphosphorylated form of the enzyme  for the PAP activity of phosphorylated WT Pah1 was ∼3-fold lower when compared with the unphosphorylated form of the WT enzyme . To resolve this issue, we subjected Pah1 to SDS-PAGE using a polyacrylamide gel containing the Phos-tag reagent, which traps and reduces the electrophoretic mobility of phosphorylated proteins . As expected .Based on the phenotypes observed and kinetics results, we questioned if the ΔRP mutation affects the endogenous phosphorylation of Pah1. This was examined by analyzing the electrophoretic mobility of the WT and ΔRP mutant enzymes upon SDS-PAGE using 5% polyacrylamide gels , 75. In Fig. 10expected , 75, WT S. cerevisiae were subject to phosphorylation site analysis by LC-MS/MS . Overall, the ΔRP mutation caused a 57% reduction in the endogenous phosphorylation of Pah1 .We sought further evidence to support the notion that the ΔRP mutation affects the endogenous phosphorylation of Pah1. Accordingly, the hyperphosphorylated WT and ΔRP mutant forms of Pah1 that were expressed and purified from i.e., RP domain) within the N-terminal IDR of the protein with predicted structure (B and C). Initial studies indicated that this region was dispensable for Pah1 function as indicated by the ability of a truncation mutant (ΔN-NCRb) to complement the temperature-sensitive phenotype of the pah1Δ mutant (A), lipid droplet formation (A), and nuclear/ER membrane morphology (A). Unlike WT Pah1, Pah1-ΔRP was also functional in the absence of the Nem1-Spo7 protein phosphatase complex. The gain-of-function attributes correlated with an increase in membrane association and PAP activity. Yet, at the same time, the ΔRP mutation caused a reduction in Pah1 abundance. As discussed above, the Nem1-Spo7 complex is essential to Pah1 function; it recruits and dephosphorylates the enzyme at the nuclear/ER membrane to facilitate the dephosphorylation of PA to produce DAG i.e., RP A, indicated by the ★) and most readily dephosphorylated by the Nem1-Spo7 complex . This finding correlated with the increase in PAP catalytic efficiency (kcat/Km) of the Pah1-ΔRP when compared with the WT enzyme (B). Indeed, the dephosphorylated/unphosphorylated form of Pah1 exhibits greater PAP activity when compared with the phosphorylated form of the enzyme . Of these sites, we speculate that the 65% reduction in the phosphorylation of Ser-602, which is the major phosphorylation site in Pah1 (B) , and we cannot rule out the possibility that loss of these sites due to the ΔRP mutation might contribute to the gain-of-function phenotypes.The sites most affected by ΔRP mutation included Ser-511, which is phosphorylated by casein kinase I ; Ser-602Fig. 2 in Pah1 B 64) an anA). OfPah1 (B) , is primPah1 (B) , 52, 53.Pah1 (B) , 50, 51,Pah1 (B) , 48, 54.Mammalian lipin proteins, which are also PAP enzymes , 77, 78,Candida albicans, Kluyveromyces lactis, Aspergillus fumigatus, and Fusarium oxysporum . Roche manufactured the protease-inhibitor cocktail tablets utilized in the study. The IgG-Sepharose, and Q-Sepharose as well as polyvinylidene difluoride membrane, and enhanced chemifluorescence substrate were acquired from GE Healthcare. Nickel-nitrilotriacetic acid agarose resin and the kits for plasmid extractions and DNA gel extractions were purchased from Qiagen. Thermo Fisher Scientific supplied the Pierce strong anion exchange columns, BODIPY 493/503, as well as the alkaline phosphatase-conjugated goat anti-rabbit IgG antibody . Bio-Rad was the supplier of molecular mass protein standards, DNA size ladders, and reagents needed for electrophoresis and Western blotting. InstantBlue (Coomassie blue) was purchased from Expedeon. Phos-tag Acrylamide AAL-107 was acquired from Wako Chemicals. Scintillation counting supplies were purchased from National Diagnostics. All radiochemicals were purchased from PerkinElemer Life Sciences. Anti-Pah1 antibody . Yeast transformants were grown in synthetic complete (SC) media lacking appropriate nutrients for plasmid maintenance. Standard methods to culture yeast cells at 30 °C were used for cell growth  at an A600 = 0.4 and incubated at 30 °C at 250 rpm until A600 = 1.0. Cells were harvested and processed immediately. Plasmid propagation was performed with E. coli strain DH5α. E. coli cells were grown at 37 °C in lysogeny broth (LB) media . Antibiotics (100 μg/ml ampicillin) were utilized to select for cells containing desired plasmids. For overexpression of Pah1 and its derivatives in E. coli cells, NiCo21(DE3)+pLysS RARE2 cells harboring pGH313 or pGS108 were grown up 1 L LB containing chloramphenicol (34 μg/ml) and ampicillin (100 μg/ml) until A600 = 1.0. Protein expression was induced by addition of isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM. Solid growth media contained 1.5% and 2% agar for E. coli and S. cerevisiae, respectively.The yeast and bacterial strains that were used in this study are listed in l growth , 96. Plan SS1026 . To exprutilized . To over14C]acetate as described previously (600 ∼0.5) or stationary (A600 ∼3.5) phase at 30 °C. Lipids were extracted following the method of Bligh and Dyer (14C]acetate.Steady-state lipid labeling was performed in yeast with PA (5000 com/nmol)  . The rea-32P]ATP .via SDS-PAGE, Phos-tag AAL-107 (20 μM) was added to otherwise standard polyacrylamide gels. For immunoblotting, protein was transferred from polyacrylamide gel to polyvinylidene difluoride (PVDF) membranes followed by probing with rabbit anti-Pah1, mouse anti-Pgk1, and rabbit anti-Cho1 antibodies at a final concentration of 2 μg/ml. Secondary antibodies used were alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies and goat anti-mouse IgG antibodies at a dilution of 1:5000. Immune complexes were detected using a chemifluorescence substrate for alkaline phosphatase. The fluorescence signals from the immunoblots were detected by Invitrogen iBright1500 imager, and the signal intensities were analyzed by the iBright Analysis Software.SDS-PAGE analyses were conducted according to standard procedures . To anali.e., expressed and purified from exponential phase pah1Δ nem1Δ cells lacking the Nem1-Spo7 protein phosphatase complex) were analyzed by LC-MS/MS at the Center for Integrative Proteomics Research at Rutgers University, NJ. The preparation and analysis were performed as previously described  with the accession number MSV000092078. All other data are contained within the manuscript or the Raw MS phosphorylation data and database search results for WT and ΔRP forms of Pah1 are deposited in the MassIVE repository (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Left ventricular non-compaction is a complex cardiomyopathy and the third largest childhood cardiomyopathy, for which limited knowledge is available. Both pathogenesis and prognosis are still under investigation. Currently, no effective treatment strategy exists to reduce its incidence or severity, and symptomatic treatment is the only clinical treatment strategy. Treatment strategies are constantly explored in clinical practice, and some progress has been made in coping with the corresponding symptoms because the prognosis of children with left ventricular non-compaction is usually poor if there are complications. In this review, we summarized and discussed the coping methods for different left ventricular non-compaction symptoms. With the advancements in diagnosis and treatment techniques, the detection rate of myocardial insufficiency in children has increased. Left ventricular non-compaction (LVNC) is a rare and complex condition in children with cardiomyopathy. It is characterized by the presence of thick and reticulated myocardial trabeculae in the ventricular cavity with deep, sunken fossae connecting to the left ventricular cavity. In recent Australian retrospective studies, the incidence of LVNC in children was reported to be approximately 0.11/100,000 .At present, LVNC is the most commonly used diagnostic method to detect the morphological characteristics of left ventricle by echocardiography. In the past cohort studies, specific echocardiographic criteria were established, including Jenni, Chin and Stollberger –7. And tIt is worth mentioning that, in recent studies, LVNC is not usually recognized and LV hypertrabeculation is perhaps more accurate . This isHowever, in pediatric cases, the use of LV hypertrabeculation in children still requires caution due to the paucity of relevant studies and the fact that, as a congenital heart disease, it is not easy to determine differences in the degree of myocardial trabecular densification in children given the relatively short time of cardiac development in children compared to adults. In this article, we tentatively follow the diagnosis of LVNC because of the inability to obtain strong support.The clinical symptoms of LVNC in children are complex. LVNC may be clinically asymptomatic or present with a variety of symptoms such as chest pain, dyspnea, and palpitations; however, three main clinical symptoms require urgent attention –12. The Generally speaking, left ventricular noncompaction is a congenital disease with unknown etiology . At presIn this review, we discuss current advances in the clinical management of the different symptoms of LVNC to further the search for more effective treatments for the various related complications and facilitate the progress of clinical research. The latest treatment strategy, indications and contraindications are compiled in Patients with LVNC can show many symptoms or no symptoms. To be sure, LVNC can lead to heart failure , 15. HowHowever, prevention and treatment of heart failure in children still require improvements. The etiology of heart failure in children, usually caused by congenital heart disease, is different from that in adults, which is usually ischemic. Therefore, the therapeutic management of heart failure in children also differs from that in adults in some respects. The onset of heart failure in children is usually associated with symptoms such as fatigue, shortness of breath, exercise intolerance, and sometimes more serious symptoms, therefore necessitating prompt and effective treatment. There are two widely accepted clinical principles of treatment: elimination of the cause and control of symptoms and disease progression , 34. TheIvabradine is a selective inhibitor of sinus node If current, which can specifically reduce the heart rate, but has no obvious effect on cardiac conduction time, myocardial contractility and ventricular repolarization . ClinicaPhase II/III clinical studies show that ivabradine can safely and effectively reduce the resting heart rate of children, and the left ventricular ejection fraction, clinical cardiac function classification and quality of life have a good improvement trend . At the Long-term use of β-blockers can improve the symptoms and quality of life of adults with heart failure, and reduce the risk of death and hospitalization. However, children's studies show that carvedilol can improve the echocardiographic parameters and serum brain natriuretic peptide (BNP) level to some extent, but it has only a tendency to improve the prognosis of clinical heart failure –49. TherSome studies demonstrate the effectiveness of using drugs such as sacubitril/valsartan and SGLT2 inhibitors for the treatment of heart failure ; howeverHowever, although these studies suggest that their use in patients with heart failure is warranted, it is unknown whether they can improve the condition of patients with LVNC. Therefore, after clinical trials and evaluation, these drugs may be promising , 53.Although pharmacological therapy can significantly improve the quality of life in children with heart failure, there is still a large proportion of children with poor prognosis owing to disease progression or other factors; therefore, these children should be considered for cardiac assist device implantation or even heart transplantation , 54. TheArrhythmias in patients with LVNC usually symptomatically manifest as weakness and palpitations , 14 and In addition, the use of implantable cardiac defibrillators can effectively prevent ventricular tachycardia and sudden death –73. ImplA direct and effective approach for the treatment of arrhythmias is catheter radiofrequency ablation , 78, whiOwing to the presence of myocardial trabecular gaps in LVNC patients, there is a high risk of thrombus formation during blood flushing , 80. StaIt is well known that anticoagulation and thrombolytic therapy are clinically applied for patients with LVNC who have thrombosis. However, the need for prophylactic anticoagulation in these patients is still controversial. Although there is a lack of prospective studies to make predictions, according to some recent studies and current medication guidelines , 82, preAnticoagulation is not necessary in asymptomatic patients or those with normal cardiac function and it even increases patient burden. In a mean follow-up of 229 patients with LVNC without AF by Fazio et al., the incidence of thromboembolic events was only 2.1% . InsteadNonetheless, anticoagulation is clinically mandatory when LVNC patients have reduced cardiac systolic function, an ejection fraction below 40%, or thromboembolism or previous atrial fibrillation –90. SincUnfortunately, in all anticoagulant therapy strategies, even within the normal treatment range, bleeding is inevitable, which is also a major complication of anticoagulant therapy , 93. TheIn addition, recent clinical case reports have shown  that a 6Gene mutations, such as those involving sarcomere and ion channel genes/proteins, can lead to LVNC . It has The implementation of gene editing technologies, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) systems , has madIn addition to the distinct symptomatic treatment described above, another potential strategy is worth exploring for patients with LVNC. It is well known that patients with symptomatic LVNC have much higher mortality and worse prognosis compared to those with an asymptomatic presentation , 114, esIn addition, genetically screening families of patients with LVNC, may also elucidate whether LVNC is hereditary and determine what changes in their family's related gene is responsible and what effects this change might bring to their relatives, which could be important for future LVNC eradication and prevention , 115. AcPatients with LVNC can potentially engage in moderate physical activity , 116; thAlthough we cannot currently predict the occurrence of LVNC and no definitive treatment is yet available, we can provide patients with a better quality of life through aggressive symptomatic and preventive treatment.Many studies have successfully identified genes associated with cardiomyopathy –125, andHowever, hopes always coexist with challenges, and the safety and effectiveness of genome editing technology cannot be guaranteed –129. CurIn conclusion, patients with LVNC of any type should be closely followed up and analyzed because of the conditions’ unknown nature and poor prognosis –134. Esp


Although softball players are often required to play in hot environments, scarce evidence is available regarding the effects of ice slurry ingestion on body temperature and pitching performance in softball pitchers in a hot environment. Thus, this study investigated the effects of ice slurry ingestion before and between innings on body temperature and softball pitching performance in a hot environment.−1 of cool fluid [9.8 ± 2.2 °C] before simulated softball games and 1.25 g·kg−1 of cool fluid between inning intervals) or an ice trial (ICE: ingestion of ice slurry [− 1.2 ± 0.1 °C] based on the same timings and doses as the CON). Participants performed both trials in an outdoor ground during the summer season .In a randomized crossover design, seven heat-acclimatized amateur softball pitchers  completed simulated softball games consisting of 15 best-effort pitches per inning for seven innings with between-pitch rest intervals of 20 s. Participants were assigned to either a control trial . No significant differences were observed between the trials in rectal temperature changes during the simulated softball game (p > 0.05). Compared to the CON, heart rate during the game was significantly decreased , and handgrip strength during the game was significantly increased  in the ICE. Ratings of perceived exertion, thermal comfort, and thermal sensation were improved in the ICE compared to those in the CON (p < 0.05). Ball velocity and pitching accuracy were not affected by ICE.Ice slurry ingestion before the simulated softball game (pre-cooling) resulted in a greater reduction in rectal temperature compared with cool fluid ingestion (Ice slurry ingestion before and between innings reduced thermal, cardiovascular, and perceptual strain. However, it did not affect softball pitching performance compared to cool fluid ingestion.The online version contains supplementary material available at 10.1186/s40101-023-00329-0. The pitcher is one of the most important positions in softball, and ball velocity is an important indicator of performance in softball pitchers. However, ball velocity generally decreases with the increase in number of pitches thrown . PossiblTc) and mean skin (sk) temperatures  to 6 [very hot]) and TC (− 6 [very uncomfortable] to 6 [very comfortable]) were measured before and after warm-up and innings . The RPEThe ball velocity was measured using a radar gun  positioned 3 m behind the home plate. Video images were recorded using an HD 60 fps camera  positioned 3 m behind the home plate. These video images were used to determine the pitching accuracy . Accuracy was determined by the same investigator. The handgrip strength of the dominant hand was measured using a handgrip dynamometer  after the warm-up, REST, and each inning.p < 0.05. Unless otherwise stated, all values are presented as the mean ± standard deviation. The Shapiro–Wilk test was used to assess the normality of distribution, and all parameters, except for the perceptual indices, were found to not differ significantly from normal. Differences in environmental variables between trials were analyzed using linear mixed models . Differences between trial, time, and trial × time for the changes in thermal, cardiovascular, and performance variables were analyzed using linear mixed models . This analysis was preferred because it allows for missing data. It also accurately models different covariate structures for repeated-measures data and models between-subject variability [Δ) in Tre during REST and the simulated softball games were calculated and analyzed using linear mixed models to adjust for potential confounding covariates . Where significance was found, the values were subsequently analyzed using the Bonferroni multiple comparison test. Differences in perceptual variables between trials were analyzed using the Wilcoxon’s matched pairs test. Cohen’s d effect size was also calculated where necessary, whereby > 2.0 was categorized as a very large effect, 1.2–2.0 as a large effect, 0.6–1.2 as a moderate effect, and 0.2–0.6 as a small effect [Statistical analysis was performed using SPSS software . Statistical significance was set at iability . Changesl effect .p = 0.539), relative humidity , and air velocity  between trials.The mean data for all participants, male participants, and female participants are presented in Supplementary Tables Tre responses are shown in Fig. Tre (p > 0.05) and sk . Tre was significantly lower in the ICE than in the CON .mentclass2pt{minimΔTre during REST and simulated softball games are shown in Fig. Tre during REST than the CON , but a main effect of the trial for HR was observed . Changes in towel-dried nude body mass, gross sweat loss, and urine-specific gravity remained comparable between the trials (p > 0.05).There was no trial × time interaction for HR  and RPE after the fourth inning . The ICE also lowered TS after the second inning  and TC after first , second , third , and seventh  innings; however, there were no significant differences between the trials.The perceptual indices between the trials are shown in Fig. p > 0.05), pitching accuracy (p > 0.05), and handgrip strength (p > 0.05). However, main effects of time on ball velocity (p < 0.001) and handgrip strength (p = 0.003) were observed. The ball velocity was significantly lower in the sixth  and seventh  innings than in the first inning. Handgrip strength was significantly lower in the sixth  and seventh  inning than at REST. There was a main effect of the trial on handgrip strength (p = 0.001). Handgrip strength was significantly higher in the ICE than in the CON .The responses for ball velocity, pitching accuracy, and handgrip strength are shown in Fig. Tre), cardiovascular (HR), and perceptual  strain. However, ice slurry ingestion had no impact on the magnitude of the ball velocity decrement with the increase in the number of pitches thrown.This study investigated the effect of ice slurry ingestion before and between innings on body temperature and softball pitching performance in a hot environment. The present findings revealed that, in accordance with our principal hypothesis, ice slurry ingestion before and between innings decreased thermal  reduced Tre compared with the ingestion of 5.0 g·kg−1 of cool fluid (− 0.35 ± 0.16 °C vs − 0.25 ± 0.13 °C). A previous study reported a 0.4 °C reduction in Tre with 7.5 g·kg−1 of ice slurry ingestion for 15 min after warm-up [Tre prior to exercise and increases heat storage capacity, which delays the influence of hyperthermia-induced fatigue [Tre without a concurrent decrease in sk, and (2) a delay in the onset of sweating during subsequent exercise [Tre increase during the early stages of subsequent exercise [Tre during simulated softball games was comparable between the trials  according to the same timing [−1) dose before simulated softball games and low (1.25 g·kg−1) doses between the innings of ice slurry ingestion, are useful for softball pitchers playing innings in a hot environment.To our knowledge, this study is the first to investigate the influence of ice slurry ingestion on  warm-up , which i fatigue . Howeverexercise . These mexercise . In the e timing . TherefoTc including brain temperature [−1 ice slurry reduced brain temperature, as measured by proton magnetic resonance spectroscopy [−1 ice slurry attenuated reductions in maximal voluntary contraction following exercise-induced hyperthermia without causing physiological changes [Tc reduction, including brain temperature, and sensory mechanisms.In the present study, neuromuscular function following ice slurry ingestion was assessed because reduced muscle strength was shown to be related to decreased ball velocity for softball pitchers . Exercisperature . A previtroscopy . A previ changes . The aut changes . The pre changes ; therefo−1 of ice slurry during game and set breaks in a tennis-simulated running test in a previous study [Tre during the simulated softball game  due to a lack of cardiovascular measurements . However, given the results of a previous study [Tre, skin blood flow, cardiac output, mean arterial pressure, or local sweat rate [−1 on a 12% incline wearing firefighter-protective clothing decreased HR by approximately 10 bpm at 5 min of the commencement of walking compared to cold fluid [The present study demonstrated that ice slurry ingestion lowered HR by approximately 10 bpm compared with cool fluid ingestion, which is consistent with the findings of a reduction in HR by ingestion of 1.25 g·kgus study . We are us study , we speceat rate . The auteat rate . The othld fluid . HoweverTre), cardiovascular (HR), and perceptual  strain, pitching performance was not affected by ice slurry ingestion. This may be due to the low thermal strain  during the simulated softball games in this study. A recent review suggested that it is easier to avoid an increase in thermal strain by employing less intense exercise protocols or protocols interspersed with moments of lower-intensity recovery periods (intermittent efforts); thus, the benefits of ice slurry ingestion are minimized [Tre during CON in the present study were lower than those observed in previous studies, which investigated changes in body temperature during a 3-h baseball practice [Although ice slurry ingestion improved thermal  trials were conducted in an outdoor ground, (2) pitchers were heat acclimatized, and (3) cool fluid (10 °C) was employed for CON. Previous studies were conducted in the laboratory; however, environmental factors, including wind velocity  and solaThis study has three limitations. First, the type of participants employed in this study  did not allow for comparison with professional athletes. Regarding the pitching accuracy, a previous study has reported that the strike rates in 480 regular season Chinese Professional Baseball League games were approximately 50% . In addi−1) and between (1.25 g·kg−1) innings improved thermal (Tre), cardiovascular (HR), and perceptual  strain compared with cool fluid ingestion. However, this strategy did not affect pitching performance.This study demonstrated that ice slurry ingestion before .  Additional file 2: Supplementary Table 2. Mean data of male participants between trials .Additional file 3: Supplementary Table 3. Mean data of female participants between trials .


H-benzimidazol-2-ones with their potential molecular targets: cholinesterases (ChE) and three types of the Gs-protein-coupled serotonin receptors (5-HTR) 5-HT6, 5-HT4 and 5-HT7 . A microplate modification of the Ellman method was used for the biochemical analysis of the inhibitory ability of the drugs towards ChE. Molecular modeling methods, such as molecular docking and molecular dynamics (MD) simulation in water and the lipid bilayer, were used to study the interaction of the compounds with ChE and 5-HTR. In vitro experiments showed that the tested compounds had moderate anticholinesterase activity. With the help of molecular modeling methods, the mechanism of interaction of the tested compounds with ChE was investigated, the binding sites were described and the structural features of the drugs that determine the strength of their anticholinesterase activity were revealed. Primary in silico evaluation showed that benzimidazole–carboxamides effectively bind to 5-HT4R and 5-HT7R. The pool of the obtained data allows us to choose N-[2-(diethylamino)ethyl]-2-oxo-3-(tert-butyl)-2,3-dihydro-1H-benzimidazole-1-carboxamide hydrochloride (compound 13) as the most promising for further experimental development.The etiology and pathogenesis of Alzheimer’s disease are multifactorial, so one of the treatment strategies is the development of the drugs that affect several targets associated with the pathogenesis of the disease. Within this roadmap, we investigated the interaction of several substituted 1,3-dihydro-2-oxo-1 From 1990 to 2019, the incidence and prevalence of Alzheimer’s disease (AD) and other dementias increased by 147.95% and 160.84%, respectively -piperazin-1-yl group significantly enhances the inhibitory ability: compound 24 is by far the strongest inhibitor of AChE, compared to the other compounds tested  enhances the inhibitory effect and changes the type of inhibition from competitive (22 vs. 27) affects neither the inhibitory activity against BChE nor the type of inhibition, while the replacement of the diethyl group in substituent R3 (compound 27) with the dimethyl group (compound 28) weakens the inhibitory effect and changes the type of inhibition from competitive to non-competitive (phenyl)methyl]-piperazin-1-yl group in the R3 substituent, has no inhibitory effect on BChE.The results of the analysis of the inhibitory ability of the selected compounds against BChE are presented in petitive . The reppetitive  to non-cpetitive . The reppetitive . Compoun24 is the best inhibitor of AChE, and compound 13 is the best inhibitor of BChE. Next, we studied the interaction of these compounds with potential targets (ChE and 5HT4R/5HT6R/5HT7R) using molecular modeling methods.Thus, according to the data of the biochemical experiment, compound 24 (the best inhibitor for AChE) as an example, we searched for possible binding sites for benzimidazolones on the surface of AChE. For this purpose, we performed the blind molecular docking of compound 24 into the AChE molecule, setting the entire protein surface as the search area. The result of the docking is shown in Using compound 24 on the surface of AChE coincide with Site S1a; therefore, according to the blind docking data, compound 24 cannot bind to Site S1a of AChE.The active site gorge connecting the catalytic active site (CAS) and the peripheral anionic site  of AChE is the binding site for competitive inhibitors (Site S1a). Depending on the structure, competitive inhibitors can either bind to CAS, or, as in the case of donepezil , occupy 24 on the AChE surface. We designated the places of their localization as sites S2a and S3a. These found binding sites topologically do not coincide with CAS; that is, they are allosteric centers. By interacting with these sites, a drug can inhibit enzyme activity non-competitively via allosteric modulation. We neglected several minor clusters  as an example, we searched for possible benzimidazolone binding sites on the surface of BChE. For this purpose, similarly to AChE, we performed the blind docking of compound 13 into the BChE molecule, setting the entire protein surface as the search area. The result of the docking is shown in Next, using compound 13 can bind at two sites. The first of them is Site S1b, which, as in the case of AChE, is the cavity connecting CAS and PAS. Compound 13 can also bind at Site S2b in the vicinity of amino acids Pro230, Val233, Thr234, Ser235, Glu238, Arg242, Val280, Tyr282, Gly283, Thr284, Leu286, Ser287, Val288, Phe357, Phe358, Pro359, Val361 and Tyr396. When AChE and BChE molecules were superimposed, it could be seen that Site S2a on the surface of AChE and Site S2b on the surface of BChE partially overlapped . As in the case of AChE, we neglected the minor cluster of conformations of compound 13 on the surface of BChE  as the control experiment. Estimated values of binding free energies (ΔG) are presented in At the next stage, we performed the targeted molecular docking of compounds 13 within sites S1a, S2a and S3a of AChE are shown in The most energetically favorable conformations of compound 13 occupies the space between CAS and PAS of AChE  are formed.Although blind docking showed that compound nhibitor . The mos24 with AChE. However, since the ΔG values for sites S2a and S3a are quite close, at the next stage, we simulated the conformational changes of the obtained complexes with the MD method; the results are described below.According to the calculated values of ΔG, Site S2a is the preferred site for the interaction of compound 13 and 24 bind with AChE more efficiently than the natural substrate.The estimated ΔG for the ACh-AChE complex is −4.7 kcal/mol, so compounds 13 in sites S1b and S2b of BChE are shown in The most energetically favorable conformations of compound 13 occupies the space between CAS and PAS of BChE, while the benzimidazole ring of the ligand forms pi–pi interaction with Tyr332, and the amino group of the drug forms a salt bridge with Glu197 and pi–cationic interaction with Trp82   . Thus, c13 and 24 with the sites of their interaction with ChE obtained with molecular docking. These complexes are compound 13 with all sites of AChE and BChE, and compound 24 with Sites S2a and S3a of AChE.Using the method of MD, we modelled the conformational changes of the complexes of compounds 13 during the simulation in three AChE ligand-binding sites is shown in 13 remains bound in Site S1a; however, during the simulation, the drug gradually moves towards the exit from the site  (S2b and/or S2b′).The movement of compound the site A. Duringthe site B. It is the site  and MD sthe site , compoun13 (the best BChE inhibitor), 24 (the best AChE inhibitor) and 15  with 5-HT4R, 5-HT6R and 5-HT7R. The results of molecular docking of the compounds into the binding sites of the studied 5-HTR are presented in At the next stage, with the help of molecular modeling methods, we performed an initial evaluation of the effectiveness of the binding of compounds 13, 24 and BIMU-8 bind in the same cavity inside 5-HT4R as serotonin does. However, due to their larger steric size, these drugs bind not at the bottom of this cavity (like serotonin) but closer to its entrance . However, ΔG values for these conformations are −6.4 kcal/mol and −3.5 kcal/mol for compound 13 and BIMU-8, respectively, which are higher than the ΔG values for the “upper” positions of the drugs. This means that the probability of finding the drugs in the “lower” position (in the bottom of the cavity) is lower than in the “upper” one. In the case of compound 24, the complexes of the drug with the “lower” site of the receptor have values ΔG > 0. It means that compound 24 cannot bind in the “lower” site at all. Compounds 13 and BIMU-8 bind to 5-HT6R at the same location as serotonin—at the bottom of the binding cavity -1H-indole-5-carboxamide (8K3)  C.4R most strongly among three types of 5-HTR. Experimental data on the effectiveness of the interaction of compounds 13 and 24 with 5-HTR have not yet been obtained. However, since these drugs are analogs of BIMU-8 (which is a specific agonist of 5-HT4R), it is quite expected that the efficiency is maximum for 5-HT4R. Compounds 13, 24 and BIMU-8 interact less efficiently with 5-HT7R and very weakly (or do not interact) with 5-HT6R.According to molecular docking data, benzimidazole–carboxamides bind to 5-HT4R with compounds BIMU-8, 13 and 24. This subtype of the serotonin receptor was chosen for two reasons: firstly, the studied drugs bind most efficiently to 5-HT4R, and secondly, the fact that these compounds and serotonin bind in different locations according to molecular docking data requires additional investigation. The complexes of the drugs with an upper site of 5-HT4R (13 with a lower site (serotonin-binding location) of 5-HT4R served as the starting conformations for MD simulations.Using the method of MD in the lipid bilayer, we tested the stability of the complexes of 5-HTf 5-HT4R A as well4R. So, we simulated only the ligands and the receptor inserted into the lipid bilayer without G-protein included in the system . The preparation of the 3D models is described in the Methods section; 13 with 5-HT4R in the lipid bilayer with a solvent (water) as an example.At the current stage of our research, we were not interested in the conformational changes of associated G-protein caused by the binding of agonists to 5-HT4R are shown in Conformational changes of the complexes of BIMU-8 with the upper and lower binding sites of 5-HT4R receptor both from the upper  [Compound orrected . Therefo5-HT2CR) .4R. For this additional experiment, we selected compounds 22 (diethyl group in substituent R3), 23 (trifluoromethyl group in substituent R1) and 27 (cyclopropyl group in substituent R2 and diethyl group in substituent R3). According to molecular docking data, these compounds bind initially in the same location as BIMU-8 (at the entrance to the cavity of the binding site); the ΔG values are −7.2, −8.1 and −7.5 kcal/mol for compounds 22, 23 and 27, respectively. According to the MD simulation, only compound 22 moves deeper towards the bottom of the binding site cavity  a high molecular weight of hybrid molecules; (b) low oral bioavailability; (c) inability to cross the blood–brain barrier; (d) action of the components of the hybrid molecule in various concentration ranges; and (e) need to optimize selectivity and affinity for different targets. It is necessary to take into account these problems and make appropriate calculations at the very first stages before proceeding to animal studies. At the same time, it should be understood that the degree of agreement between the data obtained with calculation and experimental methods is a separate issue.50 (mg/kg) for mice and rats were calculated in silico [s-protein-coupled 5-HTR . Then, based on a combination of in vitro and in silico data, we determined the structural features of the drugs that can affect their anticholinesterase and serotoninergic activity.The algorithm of the approach that we proposed (described in  and in tn silico . Based o15 and 16 as well as 22 and 23). As for the effect of diethylaminoethyl and dimethylaminoethyl substituents in the carboxamide moiety, compound 27 containing a diethylaminoethyl substituent had improved inhibitory properties compared to compound 28 containing a dimethylaminoethyl one. However, in general, the activity of 27 and 28 was weaker than that of compounds 13, 22 and 23, in which the imidazole ring contains the tert-butyl or iso-propyl substituent rather than the cyclo-propyl one. Compound 13 is the strongest inhibitor of BChE (Ki = 1.7 µM) and exhibits a significantly lower inhibitory activity against AChE (Ki = 36.1 µM). Compound 24 is the strongest inhibitor of AChE (Ki = 0.13 μM), but did not demonstrate inhibitory properties against BChE. Compound 13, with the most hydrophobic fragment at position R2 and the most mobile fragment with a less rigid skeleton at position R3, shows better inhibitory properties against BChE and lower toxicity [The biochemical analysis performed here showed the ability of the obtained structures to inhibit AChE and BChE, which expands the scope of their application as potential dual-action drugs . The compounds, in general, showed moderate inhibitory activity towards ChE and inhibited BChE more strongly than AChE . The inttoxicity  compared13 with AChE, which is consistent with our experimental data indicating that compound 13 is a competitive inhibitor of AChE. In the case of BChE, compound 13 interacts with approximately equal efficiency with both the site of competitive inhibitors S1b and allosteric Site S2b, which is inconsistent with our in vitro experiment. According to in silico data, compound 24 does not interact with Site S1a (apparently due to steric restrictions), but it effectively and rather strongly binds to allosteric Site S2a, which corresponds to the result of our biochemical experiment. Compound 24 binds to Site S1b of BChE with almost zero efficiency (and cannot compete with ACh for binding to the active site) and does not interact with Site S2b at all, which is again consistent with in vitro data on the absence of an inhibitory effect of compound 24 on BChE activity. When comparing the values of Ki obtained in vitro , while the calculated values of ΔG differ by only 0.4 kcal/mol . Compound 13 is a strictly non-competitive inhibitor of BChE .According to molecular docking and molecular dynamics data, the site of competitive inhibitors S1a is the main site of interaction of compound in vitro  with thein vitro , it can  of BChE ; however13 with Sites S1a and S1b in vitro. The interaction of 13 with the allosteric centers of ChE can also have an additional effect, which can lead to some conformational changes in the active center and decrease the efficiency of the interaction of the ligand with Sites S1a and S1b. This effect is worthy of additional computational experiments, which will expand knowledge about the mechanisms of ChE inhibition. It is the task of our next study.This effect of “overestimation” of efficiency can be explained by the fact that the interaction with the cavity of Sites S1a and S1b, located deep in the ChE globule, occurs gradually. At the first stage, a ligand anchors in PAS and then it gradually moves deep into the active site. Apparently, this additional (limiting) binding step, which is difficult to take into account when docking the ligand inside the active site, reduces the efficiency of the interaction of compound 13 binds in one region on the surface of BChE , and, during MD simulation, it passes to the neighboring region (S2b′), which was not detected with blind docking  of the allosteric site changes the surface configuration and lets the substrate move into another one (S2b′). The second identified allosteric site on the surface of AChE (S3a) cannot retain benzimidazolone molecules , its cationic group interacts with Tyr337, and the diethyl substituent of the cationic group binds even deeper in the active site gorge, closer to the catalytic triad  with the allosteric site of AChE (S2a). We assume that this may be due to their steric size. On the one hand, the compounds are bulky enough  to sink deeper to the bottom of S2a and fixate there. On the other hand, 13 and 22 are not so massive  as to occupy the entire space of the site and stay inside due to steric interactions. Moreover, 13 and 22 do not contain halogen substituents in their structure . The additional negative charge on the halogen substituents may contribute to faster drug binding in the AChE allosteric site. Thus, according to molecular docking data, the chlorine atom of compound 24 can form a halogen bond with the backbone oxygen atom of Pro537.It can also be noted that the competitive type of inhibition is characterized for BChE, while the mixed type is for AChE. In all likelihood, the explanation for this phenomenon also lies in the fact that the active center of BChE has a large steric size compared to AChE. The only exception in the case of AChE are compounds 13, 23 and 28 inhibit the enzyme in a non-competitive manner, while compounds 15, 16, 22 and 27 conduct it in a competitive manner. Compound 22 differs from 13 only in the absence of a third methyl group in substituent R2 (isopropyl in the case of 22 and tert-butyl in the case of 13). It can be assumed that the additional methyl group of compound 13 makes it bulkier, so it is more difficult for it to get into the active site of BChE . According to MD simulation, in Site S1b, compound 13 is immersed into the site by rotating the benzimidazole group (and its substituents R1 and R2) deep into the site around the immobile cationic group anchored near Trp82  have an interesting feature. They differ in substituent R3, the replacement of which changes the type of BChE inhibition from competitive to non-competitive. At the same time, the same substitution of R3 for a pair of compounds 15 and 22 (which do not contain the trifluoromethyl group) does not affect the type of inhibition. This paradox can be easily explained by referring to 13 in the site of competitive inhibitors of BChE S1b. If the benzimidazole ring contained the negatively charged trifluoromethyl substituent (as in compound 23), it would bind next to the negatively charged Asp70 of PAS, which is extremely unfavorable energetically. Therefore, compound 23 does not bind in Site S1b. In compound 16, the cationic group (which binds near Trp82) is one chemical bond further from the benzimidazole ring, so in the case of substance 16, the trifluoromethyl substituent would be located farther from PAS, and compound 16 can be a competitive inhibitor of BChE. In the case of compounds 15 and 22, the absence of the trifluoromethyl group makes the length of the molecule not a critical parameter.Compounds 4R, 5-HT6R and 5-HT7R, the information described in 4R and 5-HT7R no weaker than to ChE, i.e., at therapeutically relevant concentrations. With the help of MD simulation, we have identified the mechanism of binding of the benzimidazole–carboxamides to 5-HT4R. With the example of BIMU-8, this mechanism is shown in As for the interaction of the studied compounds with 5-HT13 and 23) to pass inside. It is interesting to note that compound 27 with cyclopropyl substituent R2  also does not reach the internal binding site within 100 ns of the simulation. We think that this result can be explained by the rigidity and lower conformational mobility of the cyclopropyl substituent.First, the compound binds on the surface of the receptor, then passes to the internal binding site. However, due to its steric size, BIMU-8 cannot sink to the very bottom of the cavity like serotonin does . The nar4R, 5-HT6R and 5-HT7R does not yet mean that it will lead to the proper conformational changes in the receptor molecule and further signal transmission. The study of the interaction of benzimidazole–carboxamides with 5-HTR in vitro, as well as a detailed conformational analysis of drug complexes with the receptors and associated G-proteins, are the goals of our future research. Nevertheless, in conclusion, we allow ourselves a little speculation regarding the ratio of calculated and experimental values. The experimentally obtained Ki values of compound 13 against AChE and BChE are 36.1 and 1.7 μM, respectively, and the calculated values of ΔG are −7.5 and −6.6 kcal/mol for Sites S1a of AChE and S2b of BChE. Thus, there is a clear discrepancy between the theoretical and experimental values, so that the question arises about the validity of the calculated values of the affinity of the substance for receptors, obtained in this work exclusively with the docking method. We explained above that the “overestimation” of the efficiency of binding compound 13 to the S1a sites of AChE may be due to the fact that the docking method does not take into account the primary sorption of compound 13 in PAS, so we suggest focusing on the following ratio: the value of ΔG of −7 kcal/mol obtained in an in silico experiment corresponds to an experimental Ki value of about 1 μM. In the case of serotonin receptors, the EC50 value for compound 15 (BIMU-8) relative to 5-HT4R is known to be 18 nM [13 and 15 are −7.8 and −8.5 kcal/mol, respectively. Thus, comparing all known data, we believe that the value of EC50 in relation to 5-HT4R for compound 13 can lie in the range of tens to hundreds of nM, and for 5-HT7R, it is an order of magnitude higher, in the range of hundreds to thousands of nM.It should be noted that the strong binding of compounds to 5-HTbe 18 nM . Estimat13 is one of the least toxic. The fact that compound 13 interacts with 5-HTR less efficiently than BIMU-8 indicates a high chance of using therapeutically relevant concentrations of compound 13 for both 5-HTR agonism and BChE inhibition.According to our data published in , compoun13 has minimal oral toxicity and high inhibitory ability against BChE, along with low inhibitory ability against AChE. The primary in silico evaluation of serotoninergic activity showed that compound 13 interacts with 5-HT4R by a mechanism similar to the known selective agonist BIMU-8; however, it is possible that due to the massive tert-butyl group, compound 13 is a less effective 5-HT4R agonist than BIMU-8. Additionally, according to molecular modeling data, compound 13 effectively interacts with the serotonin-binding center of 5-HT7R. The pool of the obtained data allows us to mark compound 13 as the most promising for further experimental investigation , which will confirm or disprove the potential of compound 13 as a drug for AD management.According to the results of our studies presented in  and here


Non-invasive recordings of gross neural activity in humans often show responses to omitted stimuli in steady trains of identical stimuli. This has been taken as evidence for the neural coding of prediction or prediction error. However, evidence for such omission responses from invasive recordings of cellular-scale responses in animal models is scarce. Here, we sought to characterise omission responses using extracellular recordings in the auditory cortex of anaesthetised rats. We profiled omission responses across local field potentials (LFP), analogue multiunit activity (AMUA), and single/multi-unit spiking activity, using stimuli that were fixed-rate trains of acoustic noise bursts where 5% of bursts were randomly omitted.Significant omission responses were observed in LFP and AMUA signals, but not in spiking activity. These omission responses had a lower amplitude and longer latency than burst-evoked sensory responses, and omission response amplitude increased as a function of the number of preceding bursts.Together, our findings show that omission responses are most robustly observed in LFP and AMUA signals (relative to spiking activity). This has implications for models of cortical processing that require many neurons to encode prediction errors in their spike output. At least since the times of von Helmholtz , predictNon-invasive human studies with steady trains of stimuli suggest that responses to omitted stimuli peak later than stimulus-evoked responses and are often reported to have lower amplitudes , but seeEarly non-invasive human studies suggested that omission responses can be observed only for relatively fast stimulus presentation rates, well above 5 Hz , which wThe attentional involvement in omission-related activity has also been found in extracellular signals recorded in the auditory cortex of trained awake macaques attending to auditory streams presented at rates of ~ 2 Hz . HoweverHere, we used penetrating microelectrodes to record local neural population activity from the auditory cortex of anaesthetised rats to trains of noise bursts at 2, 3 or 4 Hz burst rates, with noise bursts occasionally omitted at random. By combining fast presentation rates  with thWe recorded a total of 6 penetrations from 3 rats using multi-electrode eight-shank probes with 8 electrodes (channels) along each shank. Anaesthetised naive rats were exposed to trains of noise bursts presented at an isochronous rate of 2, 3 or 4 Hz, with a random subset of 5% of noise bursts omitted from each train Fig. ’ sectiont-tests. Single trials were defined as responses to single bursts (immediately preceding omitted stimuli), or expected but omitted bursts. Single-trial responses were baseline-corrected  or in the omission time window (0–250 ms relative to expected but omitted burst onset), for each channel we pooled single-trial time-average amplitude estimates across the three burst rates and entered them into one-sample rrected; ). AdditiFDR > 0.05; Fig. p < 0.05. Similarly, among 70 analysed multiunits , none showed a significant omission response  burst, suggesting that it corresponds to an offset response rather than to a true omission response. No such activity was observed for the slower burst rates (2 and 3 Hz).In contrast to LFP and AMUA signals, the analysis of spiking activity in 113 single and multi unit responses did not yield consistent omission responses. Among our 43 single units . Qualitatively, the responses to bursts presented at 3 Hz were weaker than to those presented at 2 Hz and 4 Hz, which may be explained by a form of long-term adaptation: if responses adapt to burst trains of a particular frequency as well as adjacent frequencies, 3 Hz burst responses would be expected to adapt to all rates (2–4 Hz), as opposed to 2 Hz and 4 Hz burst responses which may only adapt to their own rate and the adjacent 3 Hz. Nevertheless, the interaction between presentation rate  and stimulus (omission vs. burst) was not significant in either LFP or AMUA analysis (both p > 0.18), suggesting that the relative strength of omission responses does not depend on presentation rate.Since we only observed omission responses in the LFP and AMUA data, we further analysed these two signal types. Our first aim was to compare the latencies and amplitudes of neural responses to presented and omitted sounds Fig. B, F, and1,1176 = 8.84, p = 0.04, Fig. 1,732 = 38.21, p < 0.001, Fig. p > 0.3), suggesting that the latency of omission responses is locked to the onset of an expected but omitted noise burst, and is not modulated by presentation rate. This is consistent with omission responses rather than offset responses.There was also a significant difference in peak latency between responses evoked by omitted and presented stimuli in LFP and AMUA  but the main effects of presentation rate and the interaction effects between presentation rate and stimulus were not significant (p > 0.15). In the AMUA data, beyond the main effect of stimulus on both amplitude  = 36.612, p = 0.002) and latency , we also found a significant main effect of presentation rate on amplitude  but not on latency (p > 0.3). The interaction effects between presentation rate and stimulus were not significant (p > 0.3). Thus importantly, the latency for the LFP or AMUA omission-evoked responses had no significant dependence on presentation rate, again suggesting locking to omission onset.The same pattern of results was replicated in a control analysis, in which all channels were included  and no interaction between stimulus and presentation rate (p = 0.6405), but a main effect of presentation rate . The analysis of LFP response latency revealed no main or interaction effects (p > 0.5). Similarly, the analysis of time-shuffled AMUA data revealed no main effect of stimulus on signal amplitude (p = 0.2104) and no interaction between stimulus and presentation rate (p = 0.2974), but a main effect of presentation rate . The analysis of AMUA response latency revealed no main or interaction effects (p > 0.25). Please note that each point in the scatter plots shown in Fig. A further control analysis was performed Fig. B to confe p = 0.605, but ap < 0.001 (2 Hz); r = 0.2185, p = 0.002 (3 Hz); r = 0.5401, p < 0.001 (4 Hz). In the AMUA analysis ; r = 0.1947, p = 0.0091 (3 Hz); and r = 0.4367, p < 0.001 (4 Hz).To test whether omissions can be attributed to the same channels which show evoked responses, we analysed the correlations between the peak amplitudes of evoked and omitted responses across channels. This analysis revealed significant correlations for both types of analyses  and all presentation rates . In the LFP analysis . All significant correlations survived Bonferroni correction for multiple comparisons. In the AMUA analysis . All significant correlations survived Bonferroni correction for multiple comparisons. It should be noted that, in this analysis, omission peaks were extracted at a single-trial level , while in Figs. Next, we explored whether the size of omissions responses could depend on the number of bursts preceding it. For each burst rate, we plotted the amplitude of omission responses (averaged over channels) as a function of the number of preceding noise bursts since the previous omission. We then made a linear fit to the dependence of the amplitude on the log of the number of preceding bursts; we applied the log due to the exponential nature of adaptation effects . MeasuriTo analyse the approximate cortical depth of omission-evoked responses, we transformed the LFP data into current source density (CSD) profiles to minimise the effect of volume conduction and obtain more precise estimates of local synaptic current flow . We test1,200 = 8.53, p = 0.0046). Interestingly, we also found a significant interaction between signal type (AMUA vs. CSD) and channel depth . Given this interaction, we then compared the resulting omission response indices between response types (CSD vs. AMUA), separately for each channel depth  and in the intermediate channels , but not in the deep channels .Overall, the omission response index was significantly higher for AMUA than for CSD , but a significant difference between channel groups in the case of CSD . A post hoc analysis of the latter finding showed a significant decrease of CSD omission response indices in the superficial vs. deep channels , but not in the other pairwise comparisons (p > 0.13).When analysing AMUA and CSD as a function of depth individually, we found no significant difference between channel groups in the case of AMUA omission response indices , consistent with their interpretation as representing a predictive signal rather than an offset response. However, they are observable only in the local field potentials and analogue multiunit activity, but not in spiking activity, suggesting that they might be mediated by mechanisms other than classical spiking activity of projection neurons. In the following, we discuss the response characteristics and functional significance of omission responses.The cellular-level omission responses that we observe in the auditory cortex bear similarities with more gross non-invasive recordings of omission responses from the auditory cortex of humans. The omission response we observed had lower amplitudes and longer latencies than stimulus-evoked responses, consistent with previous non-invasive results in humans , 23, 27.There have been a few studies examining omission-related responses at a cellular level in the auditory cortex. Recent studies using calcium imaging in the auditory cortex of awake and anaesthetised naive mice have reported echo responses (following sequence termination) for long ISIs in the range of 2–4 s , 37, butWhile observing omission-related activity in naive animals suggests that it is a relatively automatic neural response, this conclusion is further reinforced by the fact that we measured neural activity from anaesthetised animals. This is consistent with previous reports that, in awake humans, omission responses can be observed under passive listening, with no attentional involvement . To the Nevertheless, the precise neural computations underlying an omission response remain to be elucidated. For instance, while an omission response may reflect a prediction error due to an unfulfilled prediction of a particular stimulus occurring at a particular time, it may also reflect a sequence-stopping response, marking the end of a temporally predictable sequence (which can only be detected after the onset of the first omitted stimulus) but independent of content-based expectations. Furthermore, at least in isochronous sequences with repeated identical standard tones, omission responses may also be explained by adaptation mechanisms without the involvement of canonical predictive processing . While pPrevious studies have occasionally interpreted omission responses as resulting from neural entrainment , 36, 37.While omission responses have been reported at very early processing stages in the visual system , invasivBased on our recordings in the auditory cortex, we observed that omission response amplitude was correlated with evoked response amplitude across channels, suggesting that the same areas that encode sounds might also signal sound omissions. Indeed, we found that channels were mostly sensitive to either sound alone  or to both presented and omitted sounds , and that very few channels (4–10%) were found to be sensitive to omissions alone. This is in contrast to a recent study  that useOne point for consideration is that while we see omission responses in the LFP and the AMUA, we do not see them in the single-unit or multi-unit activity. Although our choice of stimulus  might have influenced the observed responses, a recent study in humans has found omission responses also to entirely unpredictable stimuli , consistFirst, we must consider what the LFP and AMUA represent. LFP is considered to represent the dendritic activity of neurons, likely from summed inputs but perhaps also from dendritic processing , 40. TheOur results suggest a number of non-exclusive possibilities. (1) Omission responses are present in the spike responses of neurons but only a small fraction of them. In our sample of 113 single- and multiunits, none showed a notable emission response, indicating that this fraction, if it exists, may be small. Furthermore, if this alone is the source of omission responses, it is hard to explain the size of the omission responses in the LFP and AMUA, being about half the size of the response to the presented stimuli. One possibility is that this fraction of neurons is clustered at a particular layer or region in the auditory cortex that we did not sample sufficiently. Our sample of single- and multi-units spans the depth of the cortex and we did not see units responding to omissions at any depth. It is also possible that the fraction could grow under non-anaesthetised conditions, which should be addressed in future studies. (2) Omission responses in spikes only occur for certain stimulus conditions. For example, omission responses in the original human EEG studies were described at faster presentation rates than the ones tested here , 62, whiThis paucity of neural spiking omission responses, accompanied by a strong field potential omission response, has implications for models of the cortex. It calls into question models which require many neurons whose spike-output signals prediction or prediction error, although it could be that such models could operate for timescales or situations other than those that we examined. Our findings may be somewhat congruent with recent modelling work which proposed that prediction error or related signals are represented in the dendrites of pyramidal neurons; either in the apical dendrites –69, basaThe representation of prediction or prediction error is important for the learning or inference processes of various general hypotheses of brain function –6. UsingAuditory stimuli were delivered binaurally using custom-built in-ear headphones. The stimuli consisted of trains of broadband noise bursts presented at a fixed (isochronous) rate of 2, 3, or 4 Hz Fig. B. Each nAll experimental procedures obtained approval and licences from the UK Home Office and followed legal requirements (ASPA 1986). The subjects were three female adult Lister hooded rats weighing 225–363 g (mean = 286 g) at the time of the experiment. Rats were anaesthetised with a mixture of 0.05 ml domitor (1 mg/ml) and 0.1 ml ketamine (100 mg/ml), administered intraperitoneally. To maintain anaesthesia, rats were infused continuously with a saline solution containing 16 µg/kg/h domitor, 4 mg/kg/h ketamine and 0.5 mg/kg/h torbugesic, at a rate of 1 ml/h. Body temperature was maintained with a heating pad at 36° ± 1 °C. The depth of anaesthesia was controlled by regular testing of the absence of a toe pinch withdrawal reflex. The anaesthetised rats were placed in a stereotaxic frame with hollow ear bars set to fix the head for craniotomy. A craniotomy was performed with a centre at 4.7 mm caudal to bregma and 3.5 mm lateral to the midline. In two rats (rats 1 and 3), the craniotomy was performed over the right hemisphere; in one rat (rat 2), the craniotomy was performed over the left hemisphere.2; distance between electrodes: 0.2 mm). Anatomical coordinates were used to position the probe over the auditory cortex. The probe was then inserted into the brain at a medio-lateral orientation until all recording sites were inside the brain. First, to verify that the recording sites were driven by sound stimulation, a search stimulus consisting of broadband noise bursts was played. Next, to check that channels contained signals from neuronal populations sensitive to acoustic frequency, frequency response areas (FRAs) were measured. Following these checks, experimental stimuli were presented binaurally via headphones at approximately 80 dB SPL. The stimulus sampling rate was set to 48,828.125 Hz. Electrophysiological data were acquired at a sampling rate of 24,414.0625 Hz using a TDT system 3 recording set-up (Tucker Davis Technologies). Across rats, data were recorded from 6 penetrations . The first penetration typically targeted the coordinates of the primary auditory cortex. In rats with multiple penetrations, the consecutive experiments were performed after moving the probe towards more rostral (rat 2) or dorsal (rat 3) sites by approx. 500 μm and repeating the search stimulus and FRA recordings. The response latency of analogue multi-unit activity  with 8 shanks each with 8 equally spaced electrodes along its length, forming a square grid pattern of 8 × 8 recording sites  were filtered off-line using 7th-order two-pass Butterworth filters: a notch filter (49–51 Hz) to remove line noise, and a high-pass filter (cut-off frequency: 0.1 Hz) to remove low-frequency drifts. Data were then epoched into shorter segments, corresponding to stimulus omissions (from − 100 ms to 250 ms relative to the onset of expected but omitted stimuli) and the immediately preceding noise bursts (from − 100 ms to 250 ms relative to burst onset). Segments were selected to end at 250 ms relative to the presented or expected burst onset, since at the fastest presentation rate (4 Hz) this is when the next burst is presented. The resulting number of omission and preceding burst epochs per penetration (mean ± SD) was 89 ± 4.89 for the 2 Hz burst rate; 165.66 ± 18.78 for the 3 Hz burst rate; and 232.16 ± 10.41 for the 4 Hz burst rate.z-scoring each trace relative to the pre-stimulus baseline . To remove outliers, we calculated a standard deviation of the voltage fluctuation in each trial (SDi) and rejected trials with SDi beyond the median ± 3 SD of all SDi values. To quantify response latency, per penetration, we averaged AMUA responses across trials and burst presentation rates, and calculated the first latency relative to burst onset for which AMUA amplitude exceeded its half-maximum, relative to the pre-stimulus baseline.Short segments were analysed in three ways, to obtain measures of local field potentials (LFP), analogue multiunit activity (AMUA), and single- and multiunit spiking activity. In the LFP analysis, low-frequency signals were derived from original data by low-pass filtering each short segment using a 3rd order two-pass Butterworth filter (cut-off frequency: 75 Hz) and downsampling to 150 Hz. To avoid signals from channels with different polarity cancelling each other out, LFP traces from channels showing a negative burst-evoked peak amplitude in the period 0–250 ms after noise-burst onset (averaged across presentation rates) were sign-flipped. In the AMUA analysis, data were band-pass filtered using a 3rd order two-pass Butterworth filter between 300 and 6000 Hz . Then tht-tests. The resulting p-values were corrected for multiple comparisons using a false-discovery rate pFDR < 0.05 [To test whether single channels show noise-burst-evoked activity, single-trial amplitudes were averaged over time (from 0 to 250 ms relative to burst onset), pooled over burst rates, and  entered into a one-sample t-test for each channel. Similarly, to test whether single channels show omission-evoked activity, amplitudes were averaged over time (from 0 to 250 ms relative to expected but omitted burst onset), pooled over burst rates, and subjected to one-sample R < 0.05 . Only thOmission-evoked responses were compared with burst-evoked responses using mixed-effects modelling. In separate analyses, we compared peak amplitudes and peak latencies. In both cases, single-channel data were entered into a mixed-effects model with two fixed-effects factors  and one random-effects factor (penetration). In a control analysis, to test for the possibility that omission-related activity is due to random noise fluctuations in the post-omission time window rather than to true omission responses, we repeated the analysis described above but after shuffling single-trial data over time points , for each trial and analysed channel we extracted the peak amplitude of the omission response, averaged these peak amplitudes across channels, and correlated them with the log number of preceding noise bursts to model exponential decay . The Pean = 79 across 6 penetrations) and one putatively originating from a small population of neurons  near a recording site. Firing rate time series were calculated by binning spike times into 10 ms bins, resulting in peri-stimulus time histograms (PSTHs) sampled at 100 Hz. Only those single units and multiunits that were reliably driven by stimuli (noise bursts) were included in further analysis. In order to quantify firing reliability, we used a noise power to signal power metric [Finally, in the analysis of spiking activity, we performed offline spike sorting using the expectation–maximization algorithm Klustakwik  followedr metric , which cr metric , 83, onlp = (k + 1)/(N + 1), where k is the count of simulated PSTHs for which the root mean square (RMS) over the post-stimulus period  was greater than or equal to the RMS of the actual PSTH, and N = 1000 simulations. This procedure could yield a minimum p ≈ 0.001. The resulting p-values were corrected for multiple comparisons using a false-discovery rate pFDR < 0.05 [PSTHs were analysed to test for significant omission-evoked responses. Baseline spontaneous firing rate (SFR) was quantified as the average firing rate during the 50 ms preceding noise burst onset. To generate a null distribution, 1000 simulated PSTHs were calculated using a Poisson model assuming a constant firing rate equal to the SFR . For botR < 0.05 .t-tests (separate for each channel group) were corrected for multiple comparisons using Bonferroni correction.To test whether omission responses preferentially engage cortical activity in relatively superficial or deep channels and whether the cortical depth profile depends on the type of responses (higher-frequency/AMUA vs. lower-frequency/LFP), we analysed peak amplitudes at electrodes which yielded significant omission responses of both types (AMUA and LFP). In order to increase the depth resolution of the LFP signals, they were converted to current source density (CSD) estimates by calculating the second spatial derivative over channels. Given that each shank contained 8 electrodes, this procedure resulted in 6 CSD estimates as a function of channel depth. To make AMUA and CSD data more comparable, we therefore removed the edge channels from AMUA analysis. Data were pooled across penetrations, and electrode shanks into three groups of channels: superficial (channels 2–3 of each shank), intermediate (channels 4–5), and deep (channels 6–7). The same criteria were applied to assigning single units and multiunits  to different cortical depths, except for edge channels being included . To summarise the relative strength of omission responses, we calculated the omission response index of each channel (i.e. the peak amplitude of the omission response divided by the average peak amplitude of the omission and burst-evoked response). The resulting omission response index was always positive, lower than 1 if the omission response was weaker than the burst-evoked response, and higher than 1 if the omission response was stronger than the burst-evoked response. Omission response indices were compared between response types (AMUA vs. CSD) and channel groups  in a 2 × 3 ANOVA across channels. Post hoc two-sample