S375) located in the low-complexity domain, and used it to investigate the presence of S375 phosphorylation in a series (n = 44) of FTLD–TDP and ALS–TDP cases. Immunoblot analysis demonstrated phosphorylation of S375 to be a consistent feature of pathological TDP-43 species, including full-length and C-terminal fragments, in all FTLD–TDP subtypes examined (A–C) and in ALS–TDP. Of particular interest, however, detailed immunohistochemical analysis showed striking differences in the immunoreactivity profile of inclusions with the pTDP-43S375 antiserum among pathological subtypes. TDP-43 pathology of ALS–TDP, FTLD–TDP type B (including cases with the C9orf72 mutation), and FTLD–TDP type C all showed strong pTDP-43S375 immunoreactivity that was similar in amount and morphology to that seen with an antibody against TDP-43 phosphorylated at S409/410 used as the gold standard. In stark contrast, TDP-43 pathology in sporadic and genetic forms of FTLD–TDP type A (including cases with GRN and C9orf72 mutations) was found to be almost completely negative by pTDP-43S375 immunohistochemistry. These data suggest a subtype-specific, conformation-dependent binding of pTDP-43S375 antiserum to TDP-43 aggregates, consistent with the idea of distinct structural TDP-43 conformers  as the molecular basis for the phenotypic diversity in TDP-43 proteinopathies.Aggregation of hyperphosphorylated TDP-43 is the hallmark pathological feature of the most common molecular form of frontotemporal lobar degeneration (FTLD–TDP) and in the vast majority of cases with amyotrophic lateral sclerosis (ALS–TDP). However, most of the specific phosphorylation sites remain to be determined, and their relevance regarding pathogenicity and clinical and pathological phenotypic diversity in FTLD–TDP and ALS–TDP remains to be identified. Here, we generated a novel antibody raised against TDP-43 phosphorylated at serine 375 (pTDP-43The online version of this article (10.1007/s00401-020-02207-w) contains supplementary material, which is available to authorized users. FTLALS–TDP) . FTLD–TD defects . ALS–TDPDespite the common feature of abnormal TDP-43 accumulation, a significant pathological heterogeneity is observed among TDP-43 proteinopathies with respect to the pattern of anatomical distribution and morphology of inclusions, allowing recognition of up to five distinct pathological subtypes of FTLD–TDP –14, 16. The molecular basis explaining the clinical and pathological phenotypic variability of TDP-43 proteinopathies is so far not understood. In analogy to prion diseases and other neurodegenerative diseases such as tau- and alpha-synucleinopathies, a popular hypothesis, supported by an increasing body of evidence, is that the heterogeneity is a reflection of biochemical differences in pathological TDP-43 species that propagate in a prion-like manner with different conformers being associated with distinct seeding/toxicity activities  , 23, 29.TARDBP) either introduce or disrupt potential serine/threonine phosphorylation sites or introduce phosphomimic residues (glutamate/aspartate) [Since its initial discovery as the disease protein in FTLD–TDP and ALS–TDP, aberrant phosphorylation of TDP-43 has been recognized as one major PTM of pathological TDP-43 , 17. Thepartate)  stronglyS409/410 antibodies have facilitated the detection of TDP-43 pathology and are considered to be the most sensitive method for the immunohistological evaluation of TDP-43 pathology. However, to further dissect the potential role of phosphorylation in disease pathogenesis and the biochemical properties and potential differences of aggregated TDP-43 among TDP-43 proteinopathies, more detailed insights into additional TDP-43 phosphorylation sites are essential.TDP-43 has 41 serine, 15 threonine, and 8 tyrosine residues that might act as potential phosphorylation sites. Mass spectrometry analyses of recombinant TDP-43 in vitro phosphorylated with casein kinase 1 and of aggregated TDP-43 isolated from ALS–TDP have revealed several phosphorylated residues . HoweverTARDBP in an ALS patient, the consequences of serine 375 phosphorylation of TDP-43 have recently been analyzed, with in vitro studies demonstrating a potential role for S375 phosphorylation in regulating the nuclear-cytoplasmic distribution of TDP-43 [S375), and employed it for biochemical and detailed neuropathological analysis on a cohort of 44 sporadic and genetic ALS–TDP and FTLD–TDP cases. Phosphorylation at S375 was a consistent finding in all TDP-43 proteinopathies by immunoblot analysis. However, and most excitingly, the results of pTDP-43S375 immunohistochemistry (IHC) distinguished FTLD–TDP with type A pathology from other types of FTLD–TDP and ALS–TDP, most likely due to conformational differences of TDP-43 aggregates; thereby providing further evidence for the existence of distinct subtype-specific TDP-43 strains.Motivated by genetic findings on a p.S375G variant in f TDP-43 . To gainThe rabbit polyclonal antibody against TDP-43 phosphorylated at serine 375 was produced by Biosense/Eurogentec using their custom polyclonal antibody cross-affinity purification service. Briefly, rabbits were immunized with a phosphopeptide corresponding to amino acid 368–379 of human TDP-43: ac-EPNQAFGS(p)GNNS-C-CONH2. Phospho-specific antibodies were purified using affinity chromatography with modified and unmodified peptides. The initial validation for phospho-specificity of cross-affinity purified antibodies was performed by the company by qualitative indirect enzyme-linked immunosorbent assay (ELISA) .Human post-mortem tissues were obtained from the brain banks affiliated with the University of Tübingen, the DZNE Tübingen, and the University of British Columbia. Consent for autopsy was obtained from probands or their legal representative in accordance with local institutional review boards.C9orf72 repeat expansion, and 34 cases with clinical FTD (± ALS) including 15 with FTLD–TDP type A , 11 FTLD–TDP type B (including six with the C9orf72 repeat expansion), and 8 FTLD–TDP type C cases. Control cases included Alzheimer’s disease (n = 3), progressive supranuclear palsy (n = 2), Lewy body disease (n = 2), and neurologically healthy controls (n = 1).A total of 44 cases were selected covering a broad spectrum of clinical and genetic forms of primary TDP-43 proteinopathies Table . These iS375 was first established on two selected cases and regions with robust TDP-43 pathology  by testing different dilutions of the antiserum and antigen retrievals (boiling in EDTA or citrate-based buffers). For comparison, adjacent sections were stained with a phosphorylation-independent rabbit polyclonal TDP-43 antibody (ProteintechGroup).IHC was performed on 2–5 μm-thick sections of formalin fixed, paraffin-embedded (FFPE) tissue using the Ventana BenchMark XT automated staining system with the optiVIEW DAB detection kit (Roche). IHC for pTDP-43S375 IHC protocol (standard IHC) with antiserum dilution 1:400, and boiling in CC1 buffer  for 32 min as pretreatment. Adjacent sections were stained with a rat monoclonal pTDP-43S409/410 antibody (clone 1D3) [From all ALS–TDP and FTLD–TDP cases selected for the study, sections of different CNS regions were then stained with the established pTDP-43one 1D3) . The anaS375 antiserum, adjacent sections of selected cases were treated with 98% formic acid for 5 min before standard IHC.To investigate for conformation-depended binding of pTDP-43S409/410 antibody and the pTDP-43S375 antiserum. To investigate possible cross-reactivity of pTDP-43S375 antiserum with a subset of neurofibrillary tangles, double-label immunofluorescence was performed on AD cases using the pTDP-43S375 antiserum and a mouse monoclonal anti-pS202/205 tau antibody . Secondary antibodies were Alexa Fluor 488 conjugated anti-rabbit and Alexa Fluor 594 conjugated anti-mouse and anti-rat . 4′-6-diamidino-2-phenylindol (DAPI) was used for nuclear counterstaining.Double-label immunofluorescence was performed on select sections and cases using the pTDP-43S409/410 and pTDP-43S375 IHC included neuronal cytoplasmic inclusions (NCI), neuronal intranuclear inclusions (NII), dystrophic neurites (DN), combined threads and dot-like profiles (ThD), white matter threads, and glial cytoplasmic inclusions (GCI). Each type of pathology was graded semi-quantitatively: 0, absent; 1, mild/occasional (easy to find but not present in every medium power field); 2, moderate (at least a few in most fields); 3, severe/abundant . In sections of hypoglossal nucleus and spinal cord, a score of 0.5 was assigned when a single LMN NCI was identified in an entire tissue section. In addition, a cumulative grade was assigned for the total amount of TDP-43-immunoreactive (-ir) pathology in each anatomical region for the respective antibodies. Grading of the pathology demonstrated by pTDP-43S409/410 and pTDP-43S375 IHC was performed blinded to the clinical and genetic information and the assigned FTLD–TDP subtype. Grading of the pathology demonstrated by pTDP-43S375 IHC was performed blinded to the clinical and genetic information and the assigned FTLD–TDP subtype based on pTDP-43S409/410 stainings.The anatomical regions analyzed were precentral gyrus and LMN regions for ALS–TDP cases and hippocampus, temporal cortex, and LMN regions for FTLD–TDP cases. The different inclusion types evaluated for pTDP-43The sequential extraction of proteins with buffers of increasing stringency from fresh-frozen post-mortem frontal cortex from FTLD–TDP cases and controls was performed as described previously . BrieflyEscherichia coli BL21 Star DE3 (Invitrogen). The expressed N-terminally His-tagged TDP-43 was purified under denaturing conditions using nickel beads (Qiagen) and dialyzed against NMR buffer . For in vitro phosphorylation, 2 µg of recombinant TDP-43 were incubated with 6 U Casein Kinase 1 δ, active  at 30 °C for 40 min.Human TDP-43 cDNA was subcloned into pRSET expression vector and transformed into S375 antiserum, clone 1D3 anti-pTDP-43S409/410, mouse anti-panTDP-43 , and rabbit anti-panTDP-43 .Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblot analysis was performed using fluorescence detection and the Odyssey® CLx Imaging System (LI-COR Biosciences). Proteins were transferred to nitrocellulose membranes and blocked with Odyssey blocking buffer (LI-COR Biosciences). Antibodies were detected with CF680 or CF770 conjugated anti-rat, anti-rabbit, and anti-mouse IgG (Biotium). Primary antibodies for immunoblot analysis included the rabbit pTDP-43S375 and pTDPS409/410 IHC. Significance level was set at p < 0.05.Statistical analysis was performed with the GraphPadPrism software (version 7.01 for Windows). Wilcoxon test was used to assess for differences of pathology grades between pTDPS375 antiserum for phosphorylated TDP-43 was validated by immunoblot analysis of recombinant TDP-43 before and after in vitro phosphorylation with CK1, a kinase known to phosphorylate S375 residue [S375 antiserum, strong labeling was seen for the in vitro phosphorylated recombinant TDP-43  and the dentate gyrus of an FTLD–TDP type B case . Thus, the novel antiserum is a highly sensitive and specific tool for the further investigation of the S375 phosphorylation status in TDP-43 proteinopathies.Specificity of affinity purified pTDP-43 residue . While uS375 immunoreactivity in the spectrum of TDP-43 pathology in more detail, a cohort of 44 ALS–TDP and FTLD–TDP cases, including FTLD–TDP types A-C and those with GRN and C9orf72 mutations, was examined. Semi-quantitative assessment of distinct types of pTDP-43S375-ir inclusions in selected CNS regions was compared to the amount of TDP-43 pathology present with the pTDPS409/410 antibody as gold standard. As described in detail below, the whole spectrum of TDP-43-ir inclusions present in ALS–TDP, FTLD–TDP type B, and type C  was consistently and strongly labeled by pTDP-43S375 antiserum, whereas the vast majority of inclusions in all anatomical regions examined in FTLD–TDP type A cases  was not detected by pTDP-43S375 antiserum  including those with C9orf72 mutations. Specifically, ALS–TDP showed pTDP-43S375-ir diffuse and compact NCI as well as DN, ThD, and GCI in the spinal cord and affected cortical regions such as the precentral gyrus, hippocampus, and basal ganglia   treatment for 5 min before standard IHC. Notably, only under these harsh denaturing conditions, TDP-43 pathology became pTDP-43S375 in TDP-43 proteinopathies, we performed immunoblot analysis using protein lysates sequentially extracted from frozen brain tissues of cases and controls. The disease-specific TDP-43 signature was observed with pTDP-43S375 antiserum in the urea fraction with specific labeling of abnormal TDP-43 species including hyperphosphorylated full-length TDP-43 (band ~ 45 kDa), C-terminal fragments (bands ~ 20-25 kDa), and a high molecular smear. No band ~ 43 kDa corresponding to physiological TDP-43 was detectable in any fraction in FTLD–TDP or in controls. The biochemical signature observed for pTDP-43S375 resembles that seen with other phosphorylation-specific antibodies, i.e., pTDP-43S409/410 . In contrast, almost all TDP-43-ir inclusions  in sporadic and genetic FTLD–TDP type A cases, including cases with GRN and C9orf72 mutations, were completely negative. Notably, the immunoreactivity profile of TDP-43-ir pathology for pTDP-43S375 was closely associated and determined by the morphological pattern of TDP-43, regardless of the underlying cause.Phosphorylation-specific antibodies with those against pS409/410 as the most widely used are the most sensitive and specific markers for the detection of the complete spectrum of TDP-43 pathology by IHC , 28. WheS375 immunoreactivity by IHC in FTLD–TDP type A is simply a result of absent phosphorylation of TDP-43 at S375 in type A since our biochemical studies demonstrated the consistent presence of pTDP-43S375 in all cases including FTLD–TDP type A. Second, it is known that various factors such as fixation time, fixative, and post-mortem delay might influence the detectability of antigens in tissues. However, this possibility also seems highly unlikely, since these parameters did not differ between FTLD–TDP type A, and in other cases, no correlation was observed between the intensity of immunoreactivity and these factors in ALS–TDP and FTLD–TDP type B/C, and moreover, the same distinction of type A pathology from TDP-43 pathology in other subgroups was seen in tissues sampled at two different brain banks.Several possibilities can be excluded as explanation for the discordant staining properties of TDP-43-ir inclusions between FTLD–TDP type A and others. First, we can exclude that the lack of pTDP-43S375 antibody is buried and not accessible. The interpretation that the novel antibody recognizes TDP-43 in a conformational-dependent and type-specific manner is corroborated by the fact that FTLD–TDP type A inclusions became immunoreactive when FFPE sections were subjected to very harsh denaturing conditions by formic acid treatment for 5 min before IHC. Thus, our data provide strong evidence in support of the popular idea of biochemically distinct TDP-43 species  as the molecular basis of disease heterogeneity among TDP-43 proteinopathies. In this context, it is noteworthy that the postulated distinct biochemical property/conformation of aggregated TDP-43 recognized by our novel antibody is conserved throughout different affected CNS regions, cell types , and inclusion types  in a given subtype, indicating a common mechanism for the formation of all inclusions in an individual, regardless of the cell type and intracellular compartment, consistent with a prion-like propagation of TDP-43 aggregation in a self-templating manner.In fact, our data rather imply that TDP-43 species in type A inclusions have a different structural assembly compared to aggregates in other FTLD–TDP types and ALS–TDP in which the epitope recognized by the pTDP-43S375 to be determined by the morphological pattern of TDP-43, regardless of the underlying gene defect in genetic cases , suggests an involvement of additional factors. Our data are in line with the previous findings by Laferriere et al. in which biochemical/biophysical properties of TDP-43 aggregates were also reported to be better predicted by the pathological subtype than by the genetic cause [Evidence for biochemical heterogeneity of aggregated TDP-43 has been reported previously by description of different patterns of C-terminal fragments, protease-resistant cores, and distinct biophysical properties of aggregated TDP-43 among FTLD–TDP subtypes , 11, 29.VCP mutations and type E) and other genetically based ALS/FTLD–TDP cases  exist and it will be interesting to learn in the future how they will be classified based on pTDP-43S375 immunoreactivity [C9orf72 mutation carriers [The aim of the current study was to investigate pathological subtype-specific differences; therefore, we focused on the most common primary TDP-43 proteinopathies. However, other rarer FTLD–TDP subtypes  and human brain homogenates and lack of physiological nuclear staining in non-inclusion bearing cells by IHC suggest that phosphorylation of S375 is an abnormal event. However, we cannot rule out that phosphorylation at S375 might occur physiologically to a minor extent or under specific stress conditions. Cell culture studies with phosphomimetic TDP-43 mutations suggest that phosphorylation of S375 might modulate nuclear-cytoplasmic shuttling leading to a higher tendency to accumulate in the cytoplasm particularly under cellular stress conditions . A potenS375 antibody will not only be a valuable tool to further address the role of pS375 in disease pathogenesis, but also as an aid in subtyping of FTLD–TDP, particularly for difficult to classify cases. However, we recognize that the latter requires the validation of our discriminatory IHC approach by other research groups in their FTLD–TDP cohorts.In summary, we describe phosphorylation of TDP-43 at S375 as a consistent feature in sporadic and genetic ALS-TPD and FTLD–TDP and demonstrate that standard staining protocols using this novel antibody allows the distinction of TDP-43 pathology of FTLD–TDP type A from other FTLD–TDP types and ALS–TDP, most likely in a conformation-dependent manner, compatible with the concept of alternate TDP-43 conformers as the biochemical basis for phenotypic diversity. The newly generated pTDP-43Supplementary file 1 (PDF 605 kb)Below is the link to the electronic supplementary material.


The characterization showed that the surface morphology of five graphene-like magnetic sawdust biochars prepared with different preparation conditions were quite different. The specific surface area and pore structure increased with the increment of K2FeO4 addition. The results have shown that graphene-like magnetic sawdust biochar (1:1/900 °C) had the best removal on E2. The experimental results indicated that pseudo-first-order kinetic model and the Langmuir model could describe the adsorption process well, in which the equilibrium adsorption capacity  of 1:1/900 °C were 59.18 mg·g−1 obtained from pseudo-first-order kinetic model and the maximum adsorption capacity (qmax) of 1:1/900 °C were 133.45 mg·g−1 obtained from Langmuir model at 298K. At the same time, lower temperatures, the presence of humic acid (HA), and the presence of NaCl could be regulated to change the adsorption reaction in order to remove E2. Adsorption capacity was decreased with the increase of solution pH because pH value not only changed the surface charge of graphene-like magnetic sawdust biochar, but also affected the E2 in the water. The possible adsorption mechanism for E2 adsorption on graphene-like magnetic sawdust biochar was multifaceted, involving chemical adsorption and physical absorption, such as H-bonding, π-π interactions, micropore filling effects, and electrostatic interaction. To sum up, graphene-like magnetic sawdust biochar was found to be a promising absorbent for E2 removal from water.The occurrence of environmental endocrine disrupting chemicals (EDCs) in aquatic environments has caused extensive concern. Graphene-like magnetic sawdust biochar was synthesized using potassium ferrate (K AgriMohammad ,17. Furt2FeO4 as an activator, which possessed a positive electrochemical performance as electrode materials for supercapacitors. Zhou et al. stated that K2FeO4 was a promising reagent because it could not only improve the pore structure of the material, but also generate new active sites, such as FeO42− and Fe2O3 [2FeO4 could load the iron oxide compound on the surface of the material, which provided convenience for the magnetic ionization technology, and at the same time overcame the disadvantages of the difficulty of collecting biochar after adsorption, making the recovery of biochar a reality.There are several methods for the modification of the biochar surface, including physical or chemical activation, steam activation, and coating for the contaminants’ removal from wastewater . Among tnd Fe2O3 . In addi2FeO4 could fulfil the synchronous carbonization and graphitization for converting biomass to porous graphitic carbon biochar, which have been used as advanced electrode materials [2FeO4 to produce graphitic biomass carbon (GBC) by the simultaneous carbonization and graphitization of sawdust biochar. K2FeO4 was utilized as graphitization catalyst (Fe), activating agent (KOH) and magnetizing agent (Fe) [Sawdust is an abundant biomass waste resource everywhere and sawdust biochar has great application potential in the removal of water environmental pollutants ,25. Prepaterials ,26, cataaterials , and adsaterials . Based oent (Fe) . The syn2FeO4 dosage; (2) to analyze the surface characteristics of the adsorbent with multiple characterization methods, such as scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS); (3) to explore the adsorption performance of graphene-like sawdust biochar on E2; (4) to discuss the possible adsorption mechanism of graphene-like sawdust biochar removal E2 from water.The main research objectives of this study are: (1) to produce graphene-like sawdust biochar with different pyrolysis temperatures and different K18H24O2) was purchased from Sigma–Aldrich. K2FeO4, HA, and NaCl were obtained from Shanghai chemical corporation. Other chemicals including sodium hydroxide (NaOH), hydrochloric acid (HCl) and pH buffer solution , used to adjust the solution pH, were purchased from Shanghai Chemical Corp. Deionized water was obtained from Millipore Milli-Q water purification system. All reagents used were of analytical grade or higher and prepared with ultra-pure water.E2  and annealed temperature  were prepared to explore the excellent preparation conditions. 2.0 g BC was dispersed in aqueous 0.1 M K2FeO4 solution whose volume was 20 mL, 50 mL, and 100 mL, respectively with continuous stirring for 12 h, and then dried at 65 °C to obtain. The mass ratios of BC and K2FeO4 were 1:0.2, 1:0.5 and 1:1 respectively. Subsequently, the solid mixture  were heat-treated in a tube furnace at 900 °C for 2 h under a nitrogen atmosphere with a heating rate of 7 °C min−1. The mass ratio of 1:1 was heat-treated at a constant annealed temperature  for 2 h under a nitrogen atmosphere with a heating rate of 7 °C min−1. The obtained samples were washed with deionized water, followed by drying at 65 °C. These resultant samples were notated as 1:0.2/900 °C, 1:0.5/900 °C, 1:1/900 °C, 1:1/800 °C, and 1:1/1000 °C (mass ratio (BC: K2FeO4)/ annealed temperature). The mass ratios (BC: K2FeO4) and annealed temperature are also in Several samples with different mass ratios . The graphitic structure was analyzed by high-resolution transmission electron microscopy . The surface chemical composition was determined by X-ray photoelectron spectroscopy . Raman spectrometer was performed on a Raman Microscope  with YAG solid-state laser under wavelength of 532 nm. An X-Ray Diffractometer  was used to study the crystallinity. The magnetic properties of samples were examined using a vibratory probe sample magnetometer . The specific surface areas were determined using the Brunauer–Emmett–Teller .2FeO4) and annealed temperature on adsorbents properties were studied. Subsequently, the sample 1:1/900 °C was used for batch adsorption experiments based on the superbly adsorptive properties. In the beginning, three mass ratios  and different annealed temperature  have been used for adsorption experiments. The experiments were performed in a 100 mL conical flask with 50 mL aqueous solution containing the fixed concentration of E2 and adsorbents . More details on the selection of E2 and adsorbents concentrations were presented in The effects of mass ratios  were used to investigate the effect of salinity on adsorption behavior [−1) [Batch adsorption experiments included kinetics adsorption experiments, isothermal adsorption experiments, pH effect, ionic strength effect, and OM effect on E2 adsorption which were conducted at 25 °C with a shaking speed of 160 rpm for 24 h in the vibrational chamber excluding kinetic adsorption experiments . The soll flasks . The pH ior [−1) . The desx/Em = 280 nm/310 nm [E2 concentration was determined using an F-4500 fluorescence spectrophotometer . The emission source is a 450 w xenon lamp which collected the emission matrix spectra of 5 nm in the excitation range of 200–400 nm and 300–500 nm, respectively. Fluorescence intensity of E2 was measured at Em/310 nm . The dat2FeO4, the surface of GBC became more rough and uneven  [2 and K2O could further react with carbon over 700 °C to K. During physical and chemical activations, the carbon lattices expanded irreversibly and resulted in many micropores. Since these chemical reactions occurred at over 700 °C, the specific surface area was mainly related to the addition ratio of the K2FeO4 [2FeO4 made the microporous structure more powerful.Surface morphology and microporous structure of carbonaceous materials were characterized by SEM. The surface of the BC was relatively smooth a. With td uneven b–d. Moreon sites . During  700 °C) . Additioe K2FeO4 . More mi2·g−1. This indicated high BET surface area and total pore volume of 1:1/900 °C in the 2·g−1 and a tiny total pore volume of 0.020 cc/g. Nevertheless, the BET surface area was corresponding increase with K2FeO4 activation of the materials (1:0.2/900 °C and 1:0.5/900 °C). Definitively, the sample 1:1/900 °C demonstrated a powerfully abundant porous structure. The large accessible surface area and its suitable pore size distribution are beneficial for adsorption [The N2 adsorption-desorption isotherms and mesoporous size distribution of carbonaceous materials were presented in sorption .2FeO4 and annealed with high temperature. The graphitization was testified by Raman spectra  is generally used to reflect graphitization of samples. The ID/IG value of BC, 1:0.2/900 °C, 1:0.5/900 °C, 1:1/900 °C, 1:1/800 °C and 1:1/1000 °C were 0.81, 1.01, 1.08, 1.02, 0.98, and 1.01, respectively. The increasing of the ID/IG value indicated that the graphitization of the samples has been shown via the K2FeO4 excitation and high-temperature calcination. The 2D band of the samples 1:1/900 °C and 1:1/1000 °C also verified the formation of few-layered graphene rather than single-layer graphene [2FeO4 and annealed with high temperature. Moreover, the higher graphitization temperature was also beneficial to graphitization [The crystallization and graphitization of the samples were observed by XRD and Raman. The XRD patterns were shown in  spectra b. All saon atoms . The 2D graphene ,34,35. Ttization .The TEM images of BC and 1:1/900 °C have been compared in order to further understand the properties of the materials before and after modification. In 2FeO4. The XPS peak deconvolution for C1s, O1s, and Fe2p spectra of samples was shown in 3+ species on the surface of 1:1/900 °C [0 and Fe2+, respectively [0 was only 9.2%, while the proportion of Fe3+ and Fe2+ was 79.9% and 10.9%, respectively. The 1:1/900 °C become magnetic biochar with the presence of Fe species and produced more oxygen-containing functional groups accompanied by the oxidation of Fe3+ species. From the above characterization results, it can be confirmed that the 1:1/900 °C sample was successfully synthesized by one-step process [−1 which suggested that the adsorbent was enough to achieve solid-liquid separation by a permanent magnet. These results reflected that biochar after modification had excellent magnetic properties and could be easily separated and recycled.To get an insight into the surface chemical composition, the full survey scan XPS a spectra1/900 °C . While tectively . The pro process . SupplemAs presented in R2)  , E2 adsoace area . Moreove process ,41. In t , E2 adsIn −1, while the maximum adsorption capacity of BC was 107.78 mg·g−1 at 298 K. The results indicated that low temperature was more conducive to the removal of E2 by graphene-like sawdust biochar. The relative parameters for two models were summarized in R2), which suggested that the adsorption of E2 by graphene-like sawdust biochar occurred on a homogeneous surface were a monolayer adsorption process [n of the Freundlich adsorption isotherm model  [Kf value related to the adsorption capacity of the Freundlich adsorption isotherm model  [However, the adsorption capacity of 1:1/900 °C was obviously better than that of BC. In the  process ,39,42. Rfficult) , we coulsorbent) , it can The pH value of the solution was crucial to the adsorption process, which not only changed the surface charge of biochar, but also affected the E2 in the water. In Furthermore, to investigate the effect of ionic strength on adsorption of E2 on graphene-like sawdust biochar, different concentrations of NaCl (0.0 M–0.6 M) were added to the E2 solution, owing to the popular existence of this ionic compound in wastewater. In Natural organic matters (NOMs) have been regarded as an important factor affecting the adsorption process since they widely existed in the natural environment . They miAdsorbent recovery is an important consideration as it relates to the operating cost and the feasibility of its practical application. In Different characterizations and experiments were carried out to explore the possible E2 adsorption mechanisms onto 1:1/900 °C. In this work, the graphene-like magnetic structure of 1:1/900 °C have been observed by characterization. On the one hand, the sample 1:1/900 °C had a high surface area, abundant micropores, and high crystallinity properties. These surface structures were beneficial to E2 adsorption, while similar conclusions were also reported in ,43,48. M(1)2FeO4 was 1:1 and the second pyrolysis temperature was 900 °C, the graphene-like magnetic carbon sawdust biochar had the best adsorption capacity on E2.During the carbonization and graphitization process, when the mass ratio of BC to K(2)2FeO4 dosages and pyrolysis temperatures had an important impact on the morphology and structure of the graphene-like magnetic carbon sawdust. The surface area of graphene-like magnetic sawdust biochar was affected by the K2FeO4 dosage. The more addition, the larger the specific surface area. However, under the condition that BC and K2FeO4 was 1:1, the surface area of graphene-like magnetic sawdust biochar showed a positively obvious result.Different K(3)−1 obtained from Langmuir model. Adsorption kinetics and adsorption isotherm indicated that the E2 adsorption by graphene-like sawdust biochar occurred on a homogeneous surface were a monolayer adsorption process and controlled by multiple processes.The maximum adsorption capacity at 298 K was 133.45 mg·g(4)The pH, NaCl, and HA were crucial to the adsorption process because the adsorption capacity was affected by physical-chemical properties caused by the change of surface charge and the salting-out effect.(5)The possible adsorption mechanism for E2 adsorption on graphene-like magnetic sawdust biochar was multifaceted, involving chemical adsorption and physical absorption, such as H-bonding, π-π interactions, micropore filling effects, and electrostatic interaction.Based on the above conclusion, graphene-like magnetic sawdust biochar was found to be a promising absorbent for E2 removal from water as well as wastewater treatment, and the preparation method of graphene-like magnetic sawdust biochar was a green and feasible process. However, as a green technology, the economic viability might be a consideration and there should be some industrial viability of that process/product to be considered strictly.


We balance the interplay of the absorption, electrical and thermal conductivity of graphene via the device geometry. This approach yields remarkable device performance featuring room temperature high sensitivity  photodetectors is to funnel light into a small nanoscale active area and efficiently convert it into an electrical signal. Here, the authors couple a plasmonic antenna to hyperbolic phonon-polaritons in hexagonal-BN to highly concentrate mid-IR light into a graphene pn-junction. In fact, novel nano-optoelectronic platforms can be attained by merging HPPs functionalities with other 2D materials-based devices, such as graphene photodetectors governed by the photothermoelectric (PTE) effect. This mechanism generates a photoresponse in graphene pn-junctions19 driven by a temperature gradient and Fermi level asymmetry across the channel. Nevertheless, one of the limitations of these detectors is the low light absorption of graphene, especially for mid-IR frequencies where the photon energy becomes comparable to the typical doping level of graphene reaching the Pauli blocking regime21. This is further exacerbated by the small photoactive area of graphene pn-junctions22, limited by the cooling length of the hot carriers (0.5–1 μm)23. These limitations can be overcome by exciting HPPs and focusing them towards the photoactive area and consequently absorbing them in graphene. However, efficient exploitation of HPPs for mid-IR photodetection still remains unexplored25. In this work, we embed hBN and graphene within metallic antennas in order to couple their plasmonic interactions with HPPs and achieve highly concentrated mid-IR light on a graphene pn-junction for sensitive and fast mid-IR photodetection.Hyperbolic phonon-polaritons (HPPs) are hybridized modes of ionic oscillations and light present in polar dielectric materials, such as hexagonal-BN (hBN)λ ≈  5–7 μm  range 26 and heat diffusion away from the junction area. As a result, a symmetric electronic temperature profile Te(x) is produced in the graphene junction24, giving rise to a thermoelectric voltage VPTE ∝ S(x) ∇Te(x), where x runs along the graphene channel and S(x) represents the Seebeck coefficient which is tunable by the gates. Since  ∇Te(x) is antisymmetric, an antisymmetric S(x) is also needed to maximize the net PTE response, which is achieved by applying opposite voltages to the two gates  heat capacitance mismatch between graphene electrons and lattice, the HPP absorption does not amount to any meaningful temperature rise and thus does not contribute to the device PTE response.The absorption process in the graphene is mediated mostly by interband transitions, which mainly occur in the regions within the gap of the gates where the graphene doping is sufficiently small to avoid Pauli blocking  as shown in Fig. μW/μm2. Next, we investigate the photoresponse as a function of the two gate voltages (VL and VR), shown in Fig. 28. The highest values of photocurrent occur at pn or np configuration, specifically at VL = 1.6 V (170 meV) and VR = −0.82 V (−130 meV), which are relatively low doping levels. We note that when applying a voltage bias in the graphene channel, the photocurrent remains constant while the source-drain current increases linearly with bias  and a second peak (3.5 mA/W) around 8 μm. We also plot the simulated responsivity that is extracted from the multiphysics simulations, which considers the exact geometry of the photodetector and the whole device photoresponse  across the channel in the x direction  = Absdevice/Absair, which is the ratio between the graphene absorption incorporating all the elements of the device  to that of suspended graphene as a function of λ and the position vector r. G and responsivity are proportionally related via the electronic temperature gradient as shown in Supplementary Fig. We first identify the behavior of the resonant mechanisms, in terms of field intensity enhancement and spatial localization by studying the absorption enhancement in graphene . The value of G peaks around 6.8 μm due to the hybridization of the hBN HPPs with the antenna LSPR and to the constructive interference of the propagating HPPs occurring at x ~ ±100  nm. In fact, the different spatial patterns of G arise from the wavelength dependence of the HPP propagation angle in hBN following the equation 29). For longer wavelengths, we find a negligible G between 7 and 7.3 μm that corresponds to the hBN transverse optical (TO) phonon. We observe that the highest G values are only found for the spatially confined region (from x ~ −100 to 100 nm) where the antenna and gates overlap, which is designed to coincide with the graphene pn-junction  hybridization with the gate LSPR as presented in Supplementary Figs. For TE-polarization Fig. b, we finG, we simulate the system without the contribution of the HPPs using wavelength-independent refractive index values for the hBN  due to the complex shape of the gates and their interactions with the source–drain contacts  such that its LSPR peak fully overlaps with the hBN HPPs peak, thus obtaining 65 mA/W.To evaluate the coupling between the bow-tie antenna LSPR and the hBN HPPs, we study the responsivity as a function of the antenna length for TM-polarization as shown in Fig. λ = 6.5 μm with TE-polarization on the responsivity and NEP  by varying the width and length of the gate tip and graphene, while keeping the source-drain distance and width fixed as indicated in Fig. μm, which is larger than the experimental one (855 nm), pointing to future design and performance improvements  detector. We plot in Fig. 33), assuming the graphene thermal noise as the dominating noise source35. We emphasize that the zero-bias operation leads to low noise levels and a very low power consumption, which is given by the voltage applied to the gates. Furthermore, our design allows sensitive detection in different polarizations  of infrared imaging systems36.The sensitivity of the detector is best expressed in terms of external responsivity, which the maximum measured value is 27 mA/W . Additional tuning and wavelength sensitivity can be realized by controlling the hyperbolic material’s thickness42 or shape45.The device concept introduced in this work can be extended to detectors for other wavelengths or more specific functionalities such as hyperspectral imaging and spectroscopy. Our approach can also be combined with HPPs in other regions of the mid-IR and long-wave infrared range such as MoOμm, a total width of 2 μm and a narrow width region (tip) of 500 nm on a Si/SiO2 substrate using electron beam lithography (EBL) followed by evaporation of titanium (2 nm)/gold (30 nm). The gap between the gates is 155 nm. Afterwards, we transfer a hBN/graphene/hBN stack onto the metallic gates. We cleave and exfoliate the top and bottom hBN and the graphene onto freshly cleaned Si/SiO2 substrates, stack them following the Van der Waals assembly technique47 and release onto the gates. We then use EBL with a PMMA 950 K resist film to pattern source and drain electrodes and expose the device to a plasma of CHF3/O2 gases to partially etch the Van der Waals stack. Subsequently, we deposit side contacts of chromium (5 nm)/gold (80 nm) and lift off in acetone as described in ref. 46. We then etch the hBN-encapsulated graphene into an H-shape using a CHF3/O2 plasma and deposit 17 nm of Al2O3 using atomic layer deposition (ALD). Finally we pattern the bow-tie antenna of 2.7 μm total length (L) and with a small gap of 200 nm between its branches with EBL and deposit titanium (2 nm)/gold (80 nm). We point out that the bow-tie antenna and gate dimensions were selected based on preliminary optical simulations based on a simplified device, ignoring bow-tie antenna interactions with gates and metal electrodes, resulting in the non-optimal performance as evident from Fig. VL and VR both at the same potential), we attain 12,000 cm2 V−1 s−1 as a lower bound of the estimated mobility . We focus the mid-IR light with a reflective objective with a numerical aperture (NA) of 0.5. We measure the mid-IR power using a thermopile detector from Thorlabs placed at the sample position.We use a pulsed QCL mid-IR laser (LaserScope from Block Engineering) that is linearly polarized and has a wavelength tuning range from λ = 6.6 μm with a pulse width of 496 ns. We use a MCT as a reference detector from VIGO System model PCI-2TE-13. We measure the photoresponse using a current amplifier from FEMTO model DHPCA-100 with switchable gain and acquire the signal with an oscilloscope from Teledyne Lecroy model HDO6000.For the time-resolved measurements, we set the QCL wavelength to IPTE/Pin) × (Afocus/Adiff)49, where Pin is the power measured by the commercial power meter, Afocus is the experimental beam area at the measured wavelength and Adiff is the diffraction-limited spot size. We measure the photocurrent IPTE from the output signal of the lock-in amplifier VLIA considering 49), where ξ is the gain factor in V/A (given by the lock-in amplifier). We use the ratio Adiff/Afocus for estimating the power reaching our photodetector since Adiff is the most reasonable value one can attain when considering the detector together with an optimized focusing system  and it is widely used in the literature for comparing the performances among photodetectors49. Note that this simple geometrical scaling is not used here to predict the exact performance at a hypothetical diffraction-limited spot but rather as clear performance benchmark so to consistently compare different device architectures measured at differed focusing conditions. We usually have a ratio of Afocus/Adiff ≈ 7. This ratio is given by w0,x and w0,y we use our experimental observation that the photocurrent is linear in laser power and measure the photocurrent while scanning the device in the x-  and y-direction. Consequently, the photocurrent is described by Gaussian distributions w0,x and w0,y are the respectively obtained spot sizes  and RD the device resistance.The external responsivity is given by: Responsivity = (Supplementary Information


Furthermore, the CVD graphene substrates beneath the microstructure LEDs facilitated transfer of the LED arrays onto any foreign substrate. In this study, Cu foil was used for flexible LEDs. The flexible devices exhibited stable electroluminescence, even under severe bending conditions. Cyclic bending tests demonstrated the excellent mechanical stability and reliability of the devices.We report flexible and monolithically integrated multicolor light-emitting diode (LED) arrays using morphology-controlled growth of GaN microstructures on chemical-vapor-deposited (CVD) graphene films. As the morphology-controlled growth template of GaN microstructures, we used position-controlled ZnO nanostructure arrays with different spacings grown on graphene substrates. In particular, we investigated the effect of the growth parameters, including micropattern spacings and growth time and temperature, on the morphology of the GaN microstructures when they were coated on ZnO nanostructures on graphene substrates. By optimizing the growth parameters, both GaN microrods and micropyramids formed simultaneously on the graphene substrates. Subsequent depositions of InGaN/GaN quantum well and  Organic materials have long been studied for such applications, but higher device performance is expected when using inorganic materials due to their excellent electrical and optical characteristics as well as long-term stability and reliability4. Notably, GaN based micro-LEDs are promising candidates due to their excellent brightness and long life. However, integration of micro-LEDs for full-color displays remains to be resolved. Previous integration methods using multiple transfer steps (pick-up and drop-off) onto a flexible substrate and self-assembly limit inorganic LEDs to high-resolution displays and are neither reliable nor reproducible for mass production. The integration issue would be resolved using monolithically integrated micro-LEDs to demonstrate multicolor emissions via tuning their bandgap by varying the In composition of the InGaN/GaN quantum well (QW)7. Meanwhile, recent studies on GaN based micro- and nanostructures on chemical-vapor-deposited (CVD) graphene films have demonstrated their suitability for flexible light-emitting-device applications9. Accordingly, monolithic integration of various GaN microstructures on CVD graphene films is a critical step for the fabrication of flexible and high-resolution micro-LED displays11. Herein, we report the simultaneous growth of GaN microrods and micropyramids on CVD graphene films and the fabrication of flexible monolithically integrated multicolor micro-LEDs using the microstructures.Highly efficient and flexible multicolor micrometer-scale light-emitting diodes (micro-LEDs) are required for mobile and wearable optoelectronic devices and displays with high resolution and low power consumption2/Si substrates. For selective growth of inorganic materials on a CVD graphene film, a 300-nm-thick SiO2 layer was deposited on the CVD graphene film and patterned by conventional lithography. Then, ZnO nanotubes were selectively grown on the CVD graphene film by catalyst-free MOVPE using high-purity diethylzinc and oxygen (O2) as the reactants for Zn and O, respectively. Herein, we prepared ZnO nanostructures with different spacings of 2, 4, 6, and 8 µm and a fixed height and diameter of 3.8 and 0.6 µm, respectively magnesium as the doping source10. The typical resistivity and carrier concentration of the n-GaN were measured to be 6 × 10–3 Ω cm and 4 × 1018 electrons cm−3, respectively; those of the p-GaN were 5–10 Ω cm and 0.8–1.3 × 1017 holes cm−3 after p-type activation at 700 °C.The basic strategy for the growth of GaN layers on ZnO-nanostructured graphene films was as follows. The graphene thin films were synthesized on Cu foil and transferred onto SiOely Fig. . The grop-GaN overlayer by PI etching using O2 plasma treatment, Au/Ni layers (20/20 nm) were deposited as p-contact electrodes of the micro-LEDs. Ohmic contact was achieved by rapid thermal annealing in ambient air at 300 °C for 5 min. Subsequently, the entire device structures fabricated on the CVD graphene films were released from the SiO2/Si substrate by selective etching of the SiO2 interlayer using buffered oxide etchant. The LEDs on the CVD graphene films were transferred onto plastic and Cu foil.To fabricate the LED devices, metal electrodes were made on the GaN microstructures by evaporating metals on a patterned area. Before depositing the metal thin films, a PI layer was spin-coated on the microstructures to support the metal electrodes. Then, after exposure of the The surface morphology of the ZnO nanostructures, GaN microstructures, and the monolithically integrated micro-LEDs was investigated using field-emission scanning electron microscopy .I–V characteristics and EL spectra of the devices were measured while applying a DC voltage to the device using a Keithley 2400 source meter. The EL spectra were measured using a monochromator  and a detection system equipped with a charge-coupled device .The 2/Si substrates. Using graphene films as the substrate for the GaN microstructure growth was essential to prepare high-quality GaN microstructures and to facilitate transfer of the microstructures to arbitrary substrates for flexible device applications15. For position-controlled growth of ZnO nanotubes on the graphene films, a 300-nm-thick SiO2 layer was deposited on the CVD graphene films and patterned to 0.6-μm-diameter hole arrays with different spacings of 2, 4, 6 and 8 μm by conventional lithography. Then, ZnO nanotubes were selectively grown on the graphene films and GaN layers were coated on the ZnO nanotubes by metal–organic vapor-phase epitaxy (MOVPE)12. For simultaneous and morphology-controlled growth of GaN microstructures, we investigated the effect of the GaN growth time and the spacing between ZnO nanotubes in their arrays on the morphology of GaN microstructures when GaN was coated on the ZnO nanostructures prepared on graphene. In this study, we prepared ZnO nanostructures with different spacings of 2, 4, 6, and 8 μm and a fixed height and diameter of 3.8 and 0.6 μm, respectively  is smaller than that of the semipolar –2), growth along the semipolar facet would be energetically more favorable, thus resulting in the formation of micropyramids.Simultaneous growth of different GaN microstructures was achieved by heteroepitaxial growth of GaN layers on position-controlled ZnO nanotube arrays with different spacings prepared on graphene films. For the graphene substrate preparation, graphene thin films were synthesized on Cu foil using conventional CVD, lifted-off by wet-chemical etching, and transferred onto SiO17, so the growth rate could be significantly affected by the ZnO nanostructure spacing when it is narrower than 10 μm. When the spacing was wider than 10 µm, smaller micropyramids with poor growth selectivity were obtained (not shown). Accordingly, for larger spacing that was less than the surface diffusion length of 10 μm, more adatoms reached each microstructure, which enhanced the microstructure growth rate. The effect of surface diffusion of Ga adatoms on the surface morphology of the GaN microstructures was also confirmed by our supplementary study on the change in the surface morphology of the GaN microstructure as a function of growth temperature  layer filling, and exposure of the PI layer on the p-contact area, integrated micro-LED arrays were fabricated by depositing Ni/Au metal contact layers with an area of 50 × 50 µm2 on the top region of the as-grown LEDs. For flexible device applications, the LEDs were removed from the SiO2/Si substrate by chemical etching of the SiO2 layer using buffered oxide etchant and transferred to Cu-coated polyethylene terephthalate films. The Cu layer was also used to make an electrical connection for flexible LEDs. Figure The morphology-controlled growth of GaN microstructures enabled the fabrication of monolithically integrated GaN microstructure LEDs that displayed multicolor light emission. The basic approach for the integrated micro-LED fabrication is shown in Fig. The optical characteristics of the microrod- and micropyramid-based LEDs were further investigated by electroluminescence (EL) spectroscopy. The dominant light emission of these LEDs appeared at different wavelengths. For the microrod-based LEDs, the dominant EL emission at an applied current of 0.11 mA was observed at 506.3 nm with a shoulder peak centered at 536.9 nm. As the current level increased to 0.25 and 0.50 mA, the main EL peak shifted to 505.3 and 504.8 nm, respectively. The dominant EL peaks in Fig. The EL spectra at various current levels also indicated that the shift in the dominant EL peak with increasing current level for the microrod LEDs was much smaller than that for micropyramid LEDs. This behavior presumably resulted from the reduced quantum-confined Stark effect due to the dominant formation of QWs on nonpolar planes for microrod LEDs but on semipolar planes for micropyramid LEDs.5. Their observation of photoluminescence (PL) and cathodo-luminescence (CL) shows the similar tendency to our data demonstrating wavelength shift from blue to green and red as the structure changes from microrod to micropyramid and microdisk. In addition, Hong et al. reported the appearance of additional blue emission peak from microrod-based LED at high applied bias voltages with qualitative explanation using the field distribution model7. Based on their investigation, EL spectra in Fig. The optical characteristics of the GaN microstructure LEDs can be analyzed by comparing them with previously reported result. Robin et al. reported simultaneous growth of GaN microrod, micropyramid, and microdisk by controlling the diameter of the growth mask opening and fabricated different LEDs by subsequent deposition of InGaN/GaN QWs on those structuresThe monolithically integrated multicolor micro-LED arrays fabricated on graphene substrates demonstrated excellent mechanical stability and reliability under severe bending conditions. Figure I–V) characteristic curves of the microrod- and micropyramid-based LEDs. The I–V characteristic curves show typical rectifying behavior LEDs with a turn-on voltage of ≈ 3 V and a leakage current of 3 × 10−6 A at –3 V, originated by flows of electrons and holes from n to p and p to n layers when a forward bias voltage is applied across the junction resulting in reduction in the potential barrier.Figure Severe bending did not significantly affect the curves, which exhibited similar rectifying behavior without appreciable differences in the device parameters, such as the turn-on voltage or leakage current.Figure n-GaN microstructures on graphene films. The morphology-controlled growth of the GaN microstructures was realized by optimizing the growth parameters, including the spacing of the ZnO nanotube arrays used as the growth template, and the GaN growth time and temperature. GaN microrod and micropyramid arrays were grown on ZnO nanotubes spaced at 2 and 6 µm at 1000 °C for 10 min. The microrod and micropyramid-based LEDs emitted blue and green light, respectively. Additionally, blue shifting of the EL peaks was observed at higher applied currents from 0.1 to 0.5 mA. The EL peak shift was much smaller for microrod LEDs than for micropyramid LEDs, presumably as a result of the reduced quantum-confined Stark effect. Furthermore, the LEDs fabricated on CVD graphene films were readily transferred onto flexible substrates and exhibited reliable operation during bending. Our study investigates simultaneous growth of non-polar and semi-polar GaN microstructures on CVD graphene films and their fabrications to flexible LEDs to demonstrate multi-color light sources with a minimized blue shifting issue in a flexible form. We believe that the monolithic integrated flexible multicolor LEDs will pave the way for the development of next-generation optoelectronic devices using flexible inorganic LEDs, such as large-scale, high-resolution, and full-color flexible displays.We fabricated monolithically integrated multicolor LEDs on CVD graphene films for flexible devices, using simultaneous growth of morphology-controlled Supplementary Information.


We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants   •Several antidepressants, including SSRIs and ketamine, directly bind to TRKB•TRKB dimerization at transmembrane region forms a binding pocket for fluoxetine•Antidepressant binding to TRKB facilitates BDNF action and plasticity•Point mutation in TRKB transmembrane region blocks the effects of antidepressants Direct binding of both typical and fast-acting antidepressants to the BDNF receptor TRKB accounts for cell biological and behavioral actions of antidepressants. This mechanism directly connects antidepressant action to neuronal plasticity and may explain the slow action of typical antidepressants. Several targets for antidepressant (AD) drug action have been identified, but it is not clear how binding to these targets translates into clinical effects. Typical ADs such as tricyclic ADs (TCA), serotonin selective reuptake inhibitors (SSRI), and monoamine oxidase inhibitors (MAOI), increase the synaptic levels of monoamines by inhibiting their reuptake or metabolism, but it is unclear why their clinical effects are delayed, while the effects on monoamines are fast . The rapEssentially all ADs, including KET and R,R-HNK, increase the expression and signaling of brain-derived neurotrophic factor (BDNF) through neurotrophic tyrosine kinase receptor 2 (TRKB) . The effrd postnatal week in mice  metabolism. BDNF promotes production of CHOL in neurons  and CHOL in mice , which c in mice . Many AD in mice .These data prompted us to investigate the potential interactions between TRKB, CHOL, and ADs. We found that the TRKB transmembrane domain (TMD) senses changes in the cell membrane CHOL levels, and we elucidated its mechanism. Furthermore, we found that different AD drugs directly bind to a site formed by a dimer of TRKB TMDs, thereby facilitating cell surface expression of TRKB and promoting BDNF signaling. These data suggest that direct binding to TRKB and promotion of BDNF-mediated plasticity is a mechanism of action for AD drugs.CHOL is known to promote neuronal maturation and plasticity, but how it exerts these effects is unclear . CHOL isTRKB mostly resides in intracellular vesicles not accessible to BDNF . We founMutation of TRKB tyrosine 433, a predicted key residue in the CARC motif  to pheny439AXXXG443 (We next used atomistic molecular dynamics (MD) simulations to investigate the organization of TRKB TMD dimers . Using aAXXXG443 G–1I, a GAXXXG443 G  (i = 1.69 μM)  C, but no2.42 μM) L and S4M2.42 μM) . Binding1.69 μM) B, indica1.69 μM)  also dem1.69 μM) N, wherea1.69 μM) D, which in situ proximity ligation assay (PLA). A robust PLA signal was observed when N2A cells transfected to express TRKB were exposed to biotinylated FLX   F, similaectively C–S4G. Byectively H and S4Iectively J, which d = 1.82 μM)  E, and S,1.82 μM) K. R,R-HN1.82 μM) , 2018, b1.82 μM) H and 2I.These acute effects of ADs are not mediated by functional inhibition of acid sphingomyelinase (FIASMA) or sphingolipid metabolism , becauseDocking followed by an extensive set of 120 1-μs-long MD simulations suggested a binding site and mode for FLX in the crevice facing the extracellular side of the crossed TRKB TMD dimer A. This bAs shown above, the stable configuration of TRKB TM dimers at 20 mol % of CHOL is destabilized by increased membrane thickness at 40 mol % of CHOL G,H. RemaWe used fluorescence recovery after photobleaching (FRAP) assay in primary hippocampal neurons (DIV14) to evaluate the mobility of TRKB in neuronal spines. In neurons transfected to express GFP-tagged TRKB, the fluorescence was rapidly recovered in dendritic shafts, but not in spines after bleaching A and S6ASuper-resolution microscopy (dSTORM/TIRF) revealed that BDNF, FLX, KET, and CHOL all increased the size of clusters formed by wild-type GFP-TRKB, but not clusters of GFP-TRKB.Y433F mutants at the plasma membrane of fibroblast cell line, indicating that the increased trafficking may lead to increased cell surface expression and clustering of TRKB K–4P. TheADs are known to increase the cell surface expression of AMPA glutamate receptors and the blockade of AMPA receptors prevents the behavioral effects of KET and R,R-HNK . We confin vivo, and these effects are mediated by TRKB  in slices as well as  by TRKB . Theta-b by TRKB F. Howeve by TRKB G, consis by TRKB .Infusion of BDNF into the dentate gyrus of anesthetized rats significantly increased synaptic strength, as previously reported . HoweverADs increase the proliferation and survival of newly born dentate granule neurons . We confChronic FLX treatment reactivates critical period-like plasticity in the visual cortex of adult mice, allowing an ocular dominance (OD) shift in response to monocular deprivation, which normally only happens during a developmental critical period . In miceWe next investigated whether AD interaction with TRKB influences neuronal plasticity-dependent learning and behavior. FLX (15 mg/kg/day) for 7 days facilitated long-term memory in object location memory (OLM) test in TRKB.wt mice, but not in TRKB.Y433F mice, although the behavior of vehicle-treated TRKB.Y433F mice was similar to their vehicle-treated wild-type littermates A. A simiBDNF-TRKB signaling is known to be sufficient and necessary for the effects of ADs in the forced swimming test (FST) . FLX 15D and 6E.FLX promotes extinction of conditioned fear in a BDNF-dependent manner . The freTogether, these data demonstrate that the behavioral effects produced by different ADs were lost in TRKB.Y433F mutant mice, consistently with AD binding to TRKB. Moreover, we observed that many of these behavioral effects were also lost when ADs were co-administered with pravastatin .Further information and requests for resources and reagents should be directed to Eero Castren . This study did not generate new code.Experimental data generated in the present study is available in FigShare (in vitro assays (The cell line NIH 3T3 (mouse fibroblasts) stably transfected to express TRKB or TRKA  were used for o assays , N2A and6 cells/ml density at 5% CO2, 37°C in Neurobasal medium as described in detail  and HDR (homology directed repair) oligonucleotide (GCAAGGTCATCAGACCTGGCTCTTTCTCTCTCCTCAGGTCTTCGCCGTGGTGGTGATTGCATCTGTGGTGGGATTCTGCCTGC). Introduction of the point mutation generates a restriction site for the BbsI restriction enzyme. The targeting strategy was based on NCBI transcript NM_001025074.2.C57BL/6NTac-Ntrk2em6006(Y433F)Tac (TRKB.Y433F) mice were generated by introducing the Y433F point mutation into exon 12 of the After the administration of hormones, superovulated C57BL/6NTac females were mated with C57BL/6NTac males. One cell stage fertilized embryos were isolated from the oviducts at dpc 0.5. For microinjection, the one cell stage embryos were placed in a drop of M2 medium under mineral oil. A microinjection pipette with an approximate internal diameter of 0.5 μm (at tip) was used to inject the mixed nucleotide preparation  into the pronucleus of each embryo. After recovery, 25-35 injected one cell stage embryos were transferred to one of the oviducts of 0.5 dpc, pseudopregnant NMRI females.The genomic DNA was extracted from biopsies and analyzed by PCR. The following templates were used as controls: H2O (ctrl1), wild-type genomic DNA (ctrl2). A two primer PCR  detects the CRISPR/Cas9-induced constitutive knock-in allele as well as potential indel modifications and the unmodified wild-type allele. To distinguish indel modifications from unmodified wild-type sequences, heteroduplex analysis  has to be performed. The PCR amplicons were analyzed using a Caliper LabChip GX device. An aliquot of the PCR reaction was used to validate the presence or absence of the restriction site introduced via homology directed repair. 10U of BbsI enzyme was pipetted into the PCR reaction aliquot and incubated at the appropriate temperature. BbsI digest results in cleavage of the 493bp PCR product (HDR) in two fragments (362bp and 131bp).The PCR product of the positive animals from the restriction analysis was subcloned for further characterization of the founder animals. 12 subclones have been prescreened for the integration of the HDR oligonucleotide by restriction with BbsI. The ratio between the total number of analyzed clones and a positive restriction result allows an estimation of the HDR mosaicism of the founder animals. Subsequently, up to 4 clones were sequenced to confirm correct integration of the HDR oligo and presence of the Y433F point mutation. Finally, the TRKB.Y433F mice were crossed with C57BL6-RccHsd , in the University of Helsinki. All animals used from this strain were heterozygous for the Y433F mutation (TRKB.Y433F) or wild-type littermates, and genotyped after weaning.in vivo electrophysiology, male adult Sprague-Dawley rats were used.The SERT-KO mice [line B6.129(Cg)-Slc6atm1Kpl/J] on a C57BL/6 background were purchased from Taconic  and bred at the Biomedical Center, University of Freiburg. In every animal, tail biopsies were genotyped with PCR using primers from the Jackson Laboratory database to confirm homozygous knock-out. The SERT-KO mice showed bands at 210 bp, whereas the wild-type mice showed bands at 318 bp, which confirmed the gene disruption at the SERT locus. Finally, for in vivo experiments, fluoxetine and pravastatin were dissolved in the drinking water (0.08 or 0.1mg/l for fluoxetine and 0.08mg/l for pravastatin) and the volume consumed per cage was monitored every 3 days. R,R-HNK and ketamine were diluted in sterile saline for intraperitoneal injections.The present study used males and female mice (18-22 weeks old at the beginning of the experiments), and male Sprague-Dawley rats (15 weeks old at the beginning of the experiments). All animals used in the present study were experimentally naive before the beginning of the described experimental procedures. The animals were group housed  and randomly allocated into the experimental groups, except when the drug administration was performed in the drinking water. All behavioral experiments were conducted in at least two separated cohorts and analyzed automatically or by experienced observers blind to the treatment/genotype conditions. The number of animals in each experiment was based on previous studies from the literature cited in the correspondent section. For All experimental protocols were approved by the local ethics committees in Finland (#ESAVI/10300/04.10.07/2016), Norway (#6159) and Germany (#G-18-88).The protein simulated in this work included the transmembrane (TM) domains of a TRKB dimer (residues 427-459). For comparison, we also studied a TRKA dimer (residues 410-443). The lipid membranes considered were two-component bilayers composed of varying concentrations of palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesterol (CHOL). Drug binding studies in the case of TRKB were performed for fluoxetine (FLX). The effects of drugs on the physical properties of lipid membranes were studied for FLX, ketamine, and R,R-HNK. The Charmm36/m force field was used for proteins  and lipi-helical TM segment.The 3D structure for a segment of mouse TRKB containing its TM domain (residues 427-459) in its monomeric form was generated using the FMAP (Folding of Membrane-Associated Peptides) server . The serRosettaMP docking framework  was used2N90). The initial position of the TRKA TM region in the lipid bilayers was determined using the PPM Server  dimer was adapted from the NMR structure  in a membrane environment (POPC:CHOL = 100:0 and POPC:CHOL = 60:40) at 363 K to assess the stability of the selected TRKB TM dimers, and to obtain relaxed structures. The temperature was chosen based on an objective to foster sampling and to find the thermally most stable dimer structures. These exploratory simulations revealed that the only stable dimer model is centrally-coupled (A439-G443) in a cross-like configuration. The simulations of TRKB presented in this study were all started from conformations sampled from the simulations of these dimers. All membrane-protein systems considered in this study were constructed using CHARMM-GUI .The drug-binding pocket in the TRKB TM helix dimer was characterized by locally docking FLX to the TRKB TM dimers using RosettaScripts . 30 diffOne hundred and twenty independent MD simulations  were initiated by randomly repositioning and reorienting the drug in the vicinity of the aforementioned TRKB binding pocket by perturbing it from its docked mode. The effect of net positive charge on the stability of FLX binding was assessed by protonation of FLX in place (6 repeats × 500 ns).The details of the simulation systems reported in this study are given in As to the Y433F mutation, the results discussed in this paper refer to the heterozygous variant. Additional simulations for the homozygous variant (not discussed here) revealed similar behavior.Systems 9-14  were preSimulations of the TRKA TM region  method with Hamiltonian replica exchange  exchang were empPositional and orientational restraints in the harmonic form were employed to improve phase space overlap in the case of protein-binding calculations, except for the fully coupled state . Half haThe free energies and their statistical errors were estimated with the Multistate Bennett Acceptance Ratio (BAR) method  using AlTo test the possibility that drug-induced modulation of lipid bilayer properties could indirectly activate or contribute to the activation of TRKB, we performed additional protein-free simulations of POPC-cholesterol bilayers with varying CHOL concentration  (Systems 21-50). We used drug-to-lipid ratios of 1:20, 1:10, and 1:5.All simulations were carried out using GROMACS 2018 . The equBefore the production runs, all protein-membrane systems were equilibrated in stages with the protein heavy atoms kept restrained, at 310 K and 1 atm pressure using the Berendsen thermostat and barostat .−5 bar−1 on the xy-plane (membrane plane).All production simulations  were perThe simulation time for systems discussed in this work  was over 295 μs. Including a large variety of test simulations (∼330 μs) the total simulation time adds up to more than 625 μs.The cell viability assay was performed using CellTiterGlo  according to manufacturer’s instructions. Briefly, equal amounts of medium and the mixture of regents A and B were added to the E18 cortical cells (DIV8) cultivated in clear bottom 96-well plates and incubated for 40min. The luminescence was analyzed in a plate reader.The TRKB:pY (phosphorylated TRKB), TRKB:PLC-γ1, PLC-γ1:pTRKB.Y816 interactions and were determined by ELISA, based on the general method described in literature , with miThe surface levels of TRKB were also determined by ELISA . BrieflyGaussia princeps luciferase (GLuc) following the direct interaction of the proteins of interest in white 96-well plates. Two complementary fragments of the luciferase reporter protein were fused to the intracellular C terminus domain of TrkB or mutant TrkB(Y433F) to produce the PCA pairs GLuc1C-TRKB.wt/GLuc2C-TRKB.wt and GLuc1C-TRKB.Y433F/GLuc2C-TRKB.Y433F. Alternatively, the GLuc1C-TRKB.wt/GLuc2C-FYN or GLuc1C-TRKB.Y433F/GLuc2C-FYN pairs were used The GLuc2C-FYN construct expresses a lipid-raft enriched fragment of Src-family kinase FYN (Protein-fragment complementation assay  (PCA) wanase FYN . The GLuThe levels of total and phosphorylated TRKB at Y515 or Y816 in MG87.TRKB cells, challenged with BDNF, as well as the levels of PLCg1 and pTRKB.Y816 were measured by western-blotting .For the analysis of TRKB migration to lipid rafts, the samples from transfected N2A cells to express GFP-tagged TRKB.wt or TRKB.Y433F, challenged with BDNF, were processed to isolate detergent-resistant membrane (DRM) fractions in sucrose gradient . N2A celThe samples were centrifuged at 52000 x g for 18 hours at +4°C with a TLS-55 rotor in a Beckman Coulter XP Optima ultracentrifuge. Finally, 12 fractions per sample, collected from the top of the tube, were transferred to clean tubes, sonicated for 10 minutes in 0.25% SDS and prepared for western blotting, where the levels of GFP-tagged TRKB and flotillin-2 were analyzed.The cell-free assays (binding assays) were performed in white 96-well plates, based on the protocol used for western-blotting or ELISA . The plaMicroscale Thermophoresis - MST - experiments were performed using Monolith NT.115 (Blue/Red) instrument . The cell lysates of HEK293T cells expressing GFP-TRKB were used as a source of fluorescently labeled TRKB. HEK293T cells were transfected with GFP-fused TRKB as described and lysed 24h after transfection. To evaluate binding of fluoxetine to TRKB, cell lysates were diluted 1.5x times with MST buffer  to provide optimal level of fluorescence. The lysates of non-transfected HEK293T cells were used to evaluate background fluorescence, which appeared to be undetectable in current MST set up. Titration series of fluoxetine (0-100μM) were incubated with diluted cell lysates. The measurements were done in premium coated capillaries  using LED source with 470nm and 50% infrared-laser power at 25°C. Hill model was used to evaluate binding affinity, each data point represents mean ΔFnorm values from four independent experiments. The bound fraction of the GFP-TRKB was determined and the fitting was performed using the Hill model method incorporated by the MO Affinity Analysis v2.3 software (NanoTemper Technologies).In Situ Red Starter Kit Mouse/Goat , following manufacturer’s instructions. Briefly, N2A cells  were co-transfected by lipofectamine 2000 (ThermoFisher) to express TRKB and farnesylated EGFP and, 24h later, were treated with biotinylated fluoxetine (10 μM/15min). Cells were fixed with PFA 4% for 15 min, washed, and incubated in Duolink blocking buffer for 1h at 37°C. Then, the coverslips were incubated with streptavidin-conjugated HRP (1:5000 in Duolink antibody diluent) for 45 min at room temperature. After washing twice in Duolink washing buffer A, samples were incubated overnight at 4°C with a mix of goat anti-TRKB  and mouse anti-HRP  diluted in Duolink antibody diluent. After washing in Duolink washing buffer A, samples were incubated for 1h at 37°C with a mix of minus and plus probes diluted 1:5 in Duolink antibody diluent. Then, ligation of probes with connector oligo was done by washing the samples in washing buffer A, and incubating for 30min at 37°C with ligase (25 U/ml) diluted in the Duolink ligation buffer. Amplification was done by washing the samples in the washing buffer A, and incubating for 100min at 37°C with polymerase (125 U/ml) diluted in the amplification buffer. Coverslips were washed in buffer B, then in buffer B diluted 1:100 in milliQ water, and mounted in Duolink in situ mounting medium with DAPI. All incubation steps were done in a humidified chamber.Proximity ligation assay was done with Duolink Images were acquired in z stacks  with a Zeiss LSM 710 confocal microscope .For the fluorescence recovery after photobleaching (FRAP) experiments, E18 rat embryo hippocampal cells were plated onto glass coverslips coated with poly-L-lysine (Sigma-Aldrich) in 4-well cell culture plates at a density of 200.000 cells/well. The cultures were incubated at +37°C in serum-free Neurobasal medium  for approximately 2 weeks (DIV 14-16), renewing half of the growth media weekly. Then, they were transfected with Lipofectamine 2000 (Invitrogen) in order to overexpress GFP-tagged TRKB constructs (TRKB.WT or TRKB.Y433F). The cultures were incubated with the transfection mix  in serum-free media for 1.5 hours, followed by 3x medium washes and returned to the original growth medium after that. The cells were left at +37°C o/n to overexpress the constructs . Next daMG87.TRKB or primary cultured cortical cells were fixed with 4% paraformaldehyde in PBS, and unspecific binding sites were blocked with a blocking buffer . The MG87.TRKB cells were stained with anti-GFP, while primary cultures were incubated with anti-actin. The fluorescence was obtained following incubation with secondary antibodies. The coverslips were mounted with Dako Fluorescence Mounting Medium (#S3023). Images were acquired with Zeiss LSM710 confocal microscope (63x oil objective).For Sholl analysis of cultured hippocampal cells, confocal images were processed using FIJI ImageJ (NIH software), and the number of branches in the 80-μm range from the cell soma were counted using FIJI plugin.For direct stochastic optical reconstruction microscopy (dSTORM) MG87.TRKB cells were grown on cell view 35mm dishes with glass bottom (Greiner) coated with poly-L-lysine (Sigma). Cells were transfected to overexpress TRKB.wt or TRKB.Y433F, and 24h later treated with BDNF (10 ng/ml), fluoxetine (10 μM), cholesterol (20 μM) or ketamine (10 μM) for 15 minutes and fixed as for regular immunofluorescence imaging. Briefly, cells were fixed 24 hours post transfection with 4% PFA for 20 minutes, washed three times for 5 min with 1x PBS and incubated in blocking buffer  for 1 hour. The primary antibody against GFP  was diluted 1:100 and incubated overnight at +4°C, while the secondary antibody Alexa Fluor-conjugated goat-anti-rabbit 647 (Invitrogen) was diluted 1:500 and incubated at room temperature for 1 hour. The dishes were stored in +4°C in 1x PBS no longer than 48 hours before imaging.During imaging, cells were maintained in blinking buffer , which was freshly prepared and changed every hour. Imaging was performed in TIRF mode  to specifically image the fluorophores at the plasma membrane. A GE DeltaVision OMX SR system  equipped with an Apo N 60x/1.49 oil objective and a sCMOS camera was used to acquire the images. The 640 nm diode laser was used at 100% of power to excite the Alexa 647 fluorophores while the 405 nm laser was adjusted until approximately 50% of power in order to maintain the blinking throughout the imaging session. For each cell, 35000 frames were acquired, and the localization map of single fluorophores was reconstructed with SoftWoRx 7.0 software . For the localization map, the first 1000 frames approximately were discarded and the reconstruction parameters were maintained identical for all the samples.Reconstruction parameters: For the localization of all the blinking events in the dataset, the point spread function size factor was set at 1.550 with a local maximum factor of 0.05. Drift correction was adjusted dividing the frames into 20 groups. Tracking of the fluorophores was performed with a maximum localization precision of 100 nm without fiducial markers. The final localization map of the fluorophores identified was built with a reconstruction pixel size of 10 nm and localization precision set at 5 and 100 nm respectively for the minimum and the maximum, and a fluorophore persistence threshold set at 3 frames.The analysis of the reconstructed images was performed in Fiji. Depending on the size of picture, 5 or 10 representative regions of interest (ROIs) of 2x1 μm were blindly chosen per cell and used for particle analysis of the TRKB-positive clusters. The images acquired were from three independent experiments .In vivo electrophysiology. Intrahippocampal BDNF infusion was performed as described previously  1.5 g/kg body weight, positioned in a stereotaxic frame. The body temperature was constantly monitored and maintained at 37°C with an electric heating pad. Holes were drilled in the skull and a concentric bipolar stimulating electrode  was positioned in the angular bundle for stimulation of the medial perforant path  was glued to the infusion cannula (30 gauge). The electrode was then cut so that it extended 800 μm from the end of the cannula. The recording electrode was placed in the dentate hilus . The recording electrode was slowly lowered into the brain with 0.1 mm increment while monitoring response waveform evoked at 400 μA with biphasic rectangular test pulse of 0.033 Hz (pulse-width 0.15 ms). The tip of the infusion cannula was located in the deep stratum lacunosum-moleculare of field CA1, 800 μm above the hilar recording site and 300-400 μm above the medial perforant synapse. The infusion cannula was connected via a polyethylene (PE50) tube to a 10μl Hamilton syringe and infusion pump.Responses were allowed to stabilize for 1 hour at a stimulation intensity that produced a population spike 30% of maximum. After baseline recording for 20 min, infusion of 2μl of 1 ug/ul BDNF over 30 min at a rate of 0.067μl/min. Evoked responses were recorded for 120 min post infusion. Recorded field potentials were amplified, filtered (0.1 Hz to 10 kHz), and digitized (25 kHz). Electrophysiological data was analyzed using Datawave Technologies Software company. Changes in the fEPSP slope were expressed in percent of baseline. After recordings rats were decapitated and dentate gyri were dissected and immediately frozen on dry ice and stored at −80°C until use.Ex vivo electrophysiology. Extracellular field potentials (fEPSP) were recorded in acute slices of adult mouse hippocampus (pocampus . Adult mField excitatory postsynaptic currents (EPSPs) were recorded in an interface chamber using ACSF-filled glass microelectrodes (2-4 MΩ) positioned in the stratum radiatum of the CA1 region and amplified an Axopatch 200B amplifier. Electric stimulation (100 μsec duration) was delivered with a bipolar concentric stimulation electrode placed at the Schaffer collateral. Baseline synaptic responses were evoked every 20 s with a stimulation intensity that yielded a half-maximum response. After obtaining a 15 min stable baseline theta burst stimulation  or tetanic stimulation  was delivered and field potentials were recorded for 45 min.Input/output (I/O) curves were constructed using gradually increased stimulation intensities of 5, 10, 20, 30, 40 and 50 mV until the fEPSP reached plateau or visible population spike was seen.https://www.winltp.com) was used for data acquisition.To examine short term plasticity, we performed paired-pulse-facilitation (PPF) experiments using interpulse intervals (IPIs) of 20, 50, 100, and 200 ms and stimulation intensity evoking half-maximal fEPSP slopes were used. WinLTP  was applied on the left eye, the eyelashes were cut and the eye was sutured shut with 3 mattress sutures. At the end, antibiotic ointment  was applied on the sutured eye and Carprofen (5 mg/kg) was injected s.c. for postoperative analgesia. The monocular deprivation lasted 8 days and during that period, all animals were checked daily and resutured when showed a sign of the thread loss to prevent reopening of the eyes.Optical imaging. The saturation of haemoglobin in the primary visual cortex of the right hemisphere was measured to assess cortical activity  in order to preferentially stimulate the binocular part of the visual field. First, a green light (540 ± 20 nm) was used to illuminate the skull surface in order to find the region of interest and acquire a map of the surface vascular pattern, then the camera was focused 600 μm below the pial surface and the green light switched with a red light (625 ± 10 nm), to record the intrinsic signal. A longpass red filter (590 nm) was put between the skull surface and the camera to avoid that light from the stimulus monitor could affect the intrinsic signal acquisition. When the signal from one eye was being recorded, the other eye was protected with viscotears and covered with a patch. The continuous-periodic stimulation was synchronized with a continuous frame acquisition, frames were collected independently for each eye at a rate of 30 fps for 5 minutes and stored as a 512 × 512 pixel image, after spatial binning of the camera images.Data analysis. An analysis software package designed by Kalatsky et al. was used to perform Fourier decomposition on the cortical maps, that allowed to extract the intrinsic signal from the biological noise : this test was performed in a square arena (28cm side) with opaque walls containing cues (black stripes or spots). The adult male and female mice (18-22 weeks old) were placed for 3 consecutive days (15min per session) in the arena with two identical objects  in the same position throughout the sessions (pretest). At the test session one of the objects is moved to a different position and the number of visits (counted as sniffing or interacting with the object) to the old (A) or newly located (A’) object was determined by an observer bling to the conditions .Contextual fear conditioning: this test was modified from previous studies . BrieflyForced swimming test: animals  were placed in 5l glass beaker cylinders  for 6min. The immobility was assessed in the last 4min of the session . The watAnimals: both males and females were used in this experiment. The Bromodeoxyuridine (BrdU) (Sigma) was administered intraperitoneally at the dose of 75 mg/kg four times every 2 hr to reach 300 mg/kg total for each animal. The injection procedure was carried out at the start of the treatment . On day 21 of the drug treatment, the animals were euthanized by CO2, brains were quickly removed, followed by hippocampus dissection on ice, instantly frozen on dry ice and stored at −80°C until further use.A semiquantitative dot-blot method was performed as previously described with minor modifications . To isolThe brain samples were homogenized in 200 μL of 100% methanol  with an ultrasound sonicator  continuously for at least 30 s. The homogenates were centrifuged at 20800 g for 35 min at 4°C. The supernatant was then removed to 0.5 mL Vivaspin filter concentrators  and centrifuged at 8600 x g at 4°C for 35 min.The HPLC system for determination of the concentration of fluoxetine consisted of a solvent delivery pump  connected to an online degasser  and a ternary gradient unit (Jasco LG-1580-02), an analytical column  protected by a 0.5-mm inlet filter and thermostated by a column heater , and a fluorescence detector (Jasco Intelligent Fluorescence Detector model FP-1520). The wavelengths of the fluorescence detector were set to 290 (excitation) and 230 (emission), which have been found to be optimized for fluoxetine . The mobpost hoc test using GraphPad Prism (https://www.graphpad.com/) v6.01; or multivariate ANOVA using JASP (https://jasp-stats.org/) v.0.14.1. The F and p values are indicated in the figure legends. All experimental data used in the present study are available in FigShare under CC-BY license (https://doi.org/10.6084/m9.figshare.12698012).The data in the present study was analyzed by Student’s t test ; one- or two-way ANOVA were also applied, followed by Fisher’s LSD


The sensing behavior has been investigated in the relative humidity (RH) range of 2–99%. The observed results reveal a remarkable response and ultrafast detection even with zero applied bias (self-powered mode), with response and recovery times of ~ 10 and ~ 0.7 s, respectively. The self-powered behavior has been attributed to the inhomogeneities and the asymmetry in the contact electrodes. The highest sensitivity of ~ 5.64 × 106% can be achieved at an applied bias of 5 V. This approach of fabricating such highly responsive, self-powered and ultrafast sensors with simple device architectures will be useful for designing futuristic sensing devices.Humidity monitoring has become extremely vital in various technological fields such as environment control, biomedical engineering, and so on. Therefore, a substantial interest lies in the development of fast and highly sensitive devices with high figures of merit. Self-powered and ultrasensitive humidity sensors based on SnS In recent years, many efforts have been made to develop humidity sensors based on various transduction principles such as capacitive, resistive, acoustic and optical detection3. Nanoscale materials have been investigated for further improvement in sensing performance in terms of higher sensitivities, and faster response and recovery times. However, all these sensors require a power supply and therefore, in a device which involves a number of such sensors, power supply becomes a huge concern which can lead to incredible cost and complexity. In the current energy scenario, considerable efforts are being made in the fields of energy storage and energy generating devices11. Hence, low cost and self-powered nano-devices are desperately in demand.Humidity monitoring and control plays an essential role in maintaining the safety and quality of day-to-day life as it has applications in many fields, including environmental control, industrial production, medical and chemical monitoring, and so on16. Among these MDCs, earth abundant layered SnS2 is being extensively investigated due to its additional advantages such as non-toxicity, high stability, and most importantly, low fabrication costs17. Therefore, in recent times, SnS2 based devices have become potential and important candidates for gas and humidity sensing. Bharatula et al. have synthesized nanofilms of SnS2, which can effectively detect humidity with response and recovery times of ~ 85 and ~ 6 s, respectively18. Qin et al. have demonstrated SnS2 nanosheets prepared by chemical exfoliation, which exhibit rapid response and high sensitivity to NH3 at room temperature19. Zhang et al. have demonstrated a self-assembled SnS2 nanoflower/Zn2SnO4 hollow sphere nanohybrid device, exhibiting excellent humidity sensing properties20. Recently, Yan et al. fabricated NO2 gas sensor based on CVD grown SnS2 with ultra-high sensitivity by utilizing inherent defects under photoillumination21. Although, there are many reports on SnS2 based devices for humidity and gas sensing, the quest is always on for fabrication of devices with properties such as large area with simple designs, self-powered, ultrasensitive and room temperature humidity sensing.It is well established that metal dichalcogenides (MDCs) have been widely investigated for various applications such as photocatalysis, optoelectronics, solar cells, sensors, etc. due to their extraordinary properties and performance in nano-electromechanical devices2 thin films. Exfoliation, chemical vapour deposition (CVD), hydrothermal and solvothermal synthesis, and sputtering have been extensively used for fabricating nanofilm based devices24. However, exfoliation of nanoflakes and growth of thin films by CVD result in thin films whose sizes are in the range of few μm, thus hindering the large-scale growth optimum for industrial scale applications. Additionally, absence of ultrahigh vacuum during fabrication can often lead to unclean and contaminated interfaces. Therefore, magnetron sputtering has emerged as a perfect tool for deposition of large-scale thin films, involving ultra-high vacuum, which helps in controlling the thin film properties during deposition.There are several techniques to obtain low-dimensional SnS2 nanofilms and the effect of thickness modulation on the sensing behavior. The concept of self-powered detection lies with the presence of an internal electric field, which is strong enough to separate the charge carriers11. This phenomenon has been utilized in the designing of sensing devices. The self-powered detection can be realized by several approaches such as fabricating hybrids with piezoelectric materials, or depositing asymmetrical contact electrodes of different materials26. The most convenient way for realizing self-powered response is by introducing asymmetry in the contact electrodes of same material27, and this property has been exploited in the present study. The nanofilms have been prepared by a two-step growth process: deposition of Sn thin films on soda lime glass (SLG) substrates by dc magnetron sputtering followed by their sulfurization. Humidity sensing have been carried out at room temperature and the devices exhibit excellent detection even at zero bias, with ultrafast response. The sensitivity is found to be optimum for a film thickness of 262 nm. A possible mechanism based on charge hopping has been proposed to have an understanding about the detection phenomenon.Herein, we demonstrate self-powered humidity sensing based on SnS2 based humidity sensor. For investigation of the crystalline nature and phase purity of the thin films, X-ray diffraction (XRD) patterns were recorded and the patterns are shown in Fig. 2 with space group P3m128. The peak corresponding to (001) is found to be the most intense peak, thereby indicating z-direction oriented growth. As the thickness of the film increases, the intensity of the (001) peak becomes stronger. Moreover, the hump observed ~ 25° corresponds to the amorphous nature of SLG and decreases with increasing film thickness. The crystalline nature of the films is confirmed by Raman spectroscopy and the spectra have been presented in Fig. 1g (~ 312 cm−1) and Eg (~ 224 cm−1) can be clearly seen for all the three samples, which correspond to the vertical out of plane vibrations and the non-degenerate in-plane vibrations, respectively18. Thus, the formation of crystalline SnS2 thin films on SLG substrate is confirmed.Figure 2 thin films are shown in Fig. The surface morphology and the cross-sectional scanning electron microscopy (SEM) images of the SnS2 environment)–99% (highly humid atmosphere) with the help of a lab-built setup  curves of the devices under the influence of different biasing conditions (0–2 V) were recorded to examine the response/recovery times and the cyclic stability of the devices. It was observed that the current increases as the humidity level is increased and gradually saturates. However, there was a rapid drop in the current as the humidity is lowered by flushing the chamber with dry nitrogen. The sensors were tested with different biases and similar kind of behavior was observed.The humidity sensing behavior (at room temperature) of the devices has been investigated in the range of 2%  graphs of the three devices at two different humidity levels (85% and 99% RH). The I–V characteristics of all the devices have been shown with respect to the same values of the negative and positive voltage  and recovery (tf) times have been evaluated by estimating the difference in time for the signal to rise or fall from 10 to 90% or 90% to 10% of the final value, respectively and have been presented in Table 2 films may not be much favorable at room temperature, therefore leading to a larger response time. Moreover, limited by our experimental setup, the introduction of humidity into the test chamber is very gradual. Therefore, the saturation of humidity (99% RH) takes a considerable time, which is reflected by the response times of our devices. On the other hand, during the recovery time, water molecules might be easily getting desorbed due to lower adsorption and the external flushing by dry nitrogen gas, resulting in a very fast recovery time18.The response  roughness of the samples  leads to the slower recovery as compared to the other samples. Figure + ions) cannot move freely in the immobile chemisorbed and the first physisorbed layers of water molecules3. With increase in humidity levels (> 70% RH), the H+ ions can move freely in higher-level layers of physisorbed water molecules according to the Grotthuss reaction  by gently exhaling on the sample, kept ~ 4.02 nanofilms devices is extremely well compared to other 2D materials-based humidity sensors reported in literature41. A comparison of the sensing characteristics of our devices with other reported devices has been presented in Table 2-based ultrafast and self-powered humidity sensors are robust and low cost that might open routes for novel applications such as in breath monitoring. The self-powered behavior of our sensors can be extremely useful in systems which involve an array of such sensors, and therefore, the operational cost and the heavy circuitry becomes a big concern in the current scenario of energy crisis. Our findings may also enable nano-porous thin film-based sensors to exploit the unique advantages unveiled from the ultrathin nature of the constituent sheets and the facile synthesis processes, suitable for applications in fields such as consumer’s electronics, environmental and health monitoring.The performance of these SnS2 thin films on soda lime glass substrates by a two-step synthesis process—dc sputtering of Sn films followed by their sulfurization. XRD and Raman spectra confirmed the hexagonal crystal structure and SEM–EDS micrographs confirmed homogeneity and uniformity of SnS2 films over a large area. Humidity sensing measurements of SnS2 thin film-based devices of different thicknesses exhibit a highly responsive detection at 0 V for all the devices, showing self-powered nature of the devices. Temporal response was done at different bias voltages (0–2 V) and it is observed that the devices show high electrical stability and repeatability in ambient conditions. From the transient response curves, ultrafast response and recovery times of ~ 10 s and ~ 0.7 s, respectively, have been estimated for the self-powered detection of the sensor with optimum film thickness. Furthermore, a maximum sensitivity of ~ 5.64 × 106% in 99% RH at an applied bias of 5 V can be achieved. These results could pave a way for designing self-powered and ultrafast humidity sensors with simple architectures through easy approaches.We have successfully fabricated 2D SnS42. All the deposition parameters were optimized to obtain best conditions for continuous thin film and pure phase.SLG substrates were first sonicated for 15 min in iso-propyl alcohol followed by sonication in deionized water for 10 min. The substrates were then purged with nitrogen gas to remove any residuals before being loaded into the sputtering chamber (Excel Instruments). The thin films were prepared by a two-step process which involved: (i) deposition of Sn thin films on SLG by dc magnetron sputtering of Sn target (3 inch in diameter) at 45 W—15 min (sample A), 5 min (sample B), 1 min (sample C) and (ii) sulfurization  at 450 °C for 1 h in a tube furnace with continuous Ar gas flow. The sulfurization process has been explained in our previous reportThe crystal structure of the prepared films was examined by X-ray diffraction (XRD)—X’pert-PRO PANalytical with Cu Kα radiations (1.5418 Å). The surface morphology and compositional analysis of the films were determined using a scanning electron microscope (SEM)—Ultra55 FE-SEM Karl Zeiss—EDS. Thickness of the films were measured by cross-sectional SEM. Atomic force microscopy (AFM) was carried out using A.P.E. Research A100-AFM. Raman analysis was done using LabRAM HR equipped with a 532 nm laser.2 films to make metal–semiconductor–metal type contacts. A very simple computer-controlled setup which consisted of a test chamber, a mass flow controller, a source for humid air, and a data acquisition system was employed for humidity sensing measurements. During measurements, humid air was obtained by passing nitrogen gas through water and the flow was controlled with a mass flow controller. The conductivity of the samples was then measured with a Keithley 2400 source measuring unit, in different RH conditions.For humidity sensing measurements, 120 nm thick Cr/Au (10 nm/110 nm) contacts were deposited on the SnSSupplementary Information.