The anti-inflammation associated properties of methanolic extracts of cooked leaves were evaluated using four in vitro biological assays, namely, hemolysis inhibition, proteinase inhibition, protein denaturation inhibition, and lipoxygenase inhibition. Results revealed that the frying of all the tested leafy vegetables had reduced the inhibition abilities of protein denaturation, hemolysis, proteinase, and lipoxygenase activities when compared with other food preparation methods. Steaming significantly increased the protein denaturation and hemolysis inhibition in O. zeylanica and P. edulis. Steaming of leaves increased inhibition activity of protein denaturation in G. lactiferum (by 44.8%) and P. edulis (by 44%); hemolysis in C. asiatica, C. auriculata, and S. grandiflora; lipoxygenase inhibition ability in P. edulis (by 50%), C. asiatica (by 400%), and C. auriculata leaves (by 250%); proteinase inhibition in C. auriculata (100%) when compared with that of raw leaves. In general, steaming and boiling in contrast to frying protect the health-promoting properties of the leafy vegetables.The aim of the present study was to evaluate the effect of cooking  on anti-inflammation associated properties  Cassia auriculata, Gymnema lactiferum, Olax zeylanica, Sesbania grnadiflora, Passiflora edulis, and Centella asiatica are reported to possess higher antioxidant activities, as described in Gunathilake and Ranaweera [Many degenerative human diseases, such as cancer, inflammation, and cardiovascular diseases have been recognized as a consequence of free radical damage.  Inflammaanaweera . Althougp < 0.05) in steamed leaves of O. zeylanica (by 44.8%) and P. edulis (by 44%) when compared with that of their fresh leaves. Interestingly, frying of all the tested leafy vegetables resulted in the lowest protein denaturation inhibition ability when compared with other processing methods. Boiled leaves of O. zeylanica, C. auriculata, S. grandiflora, and G. lactiferum have shown significantly lower (p < 0.05) inhibition ability when compared with their fresh leaves, whereas boiled leaves of C. asiatica, and P. edulis have shown a significantly higher (p < 0.05) protein denaturation inhibition ability. Significantly lower protein denaturation inhibition ability was observed in all of the cooked leaves of C. auriculata, S. grandiflora, and G. lactiferum when compared with their fresh leaves. In a study, methanol extract of whole plant of Oxalis corniculata Linn  was assessed by Sakat et al. [50 values of 288.04 ± 2.78 µg/mL, respectively. Based on another study, a flavonoid-rich fraction of M. myristica have shown an albumen denaturation in a dose-dependent manner and the maximum inhibition of denaturation of albumin was found to around 75.38% ± 0.56% at 350 μg/mL: IC50 value of 258 μg/mL, while a standard anti-inflammatory drug (aspirin) showed a maximum inhibition of 98.41% ± 0.13% at the same concentration [Denaturation of proteins is a well-documented cause of inflammation . As partt et al.  for its ntration . Howeverntration . Accordintration , these nAccording to Chippada and co-authors , lysosomp < 0.05) the hemolysis inhibition ability of O. zeylanica, C. auriculata, S. grandiflora, G. lactiferum, P edulis, and C. asiatica by 4.0%, 5.5%, 6.0%, 6.2%, 7.0%, and 5.1%, respectively. Boiled leaves of C. asiatica, C. auriculata, and S. grandiflora have shown a significantly higher (p < 0.05) hemolysis inhibition ability hen compared with that of raw and their other cooked leaves. Steamed leaves of O. zeylanica, P. edulis, and G. lactiferum have shown the similar or higher hemolytic inhibition ability than that of their raw and other cooked leaves. Steamed and boiled leaves of S. grandiflora showed significantly higher (p < 0.05) hemolytic inhibition ability when compared with that of its raw leaves. In a previous study, the methanolic extract of the whole plant of Oxalis corniculata Linn has been shown the red blood cells membrane stabilization with the IC50 values of 467.1 ± 9.6 µg/mL [The heat-induced hemolysis inhibition abilities of raw and cooked leaf samples are shown in Cymbopogon giganteus from Benin has potential to be used as an anti-inflammatory agent towards lipoxygenase inhibition [p < 0.05) in O. zeylanica, P. edulis, C. asiatica, and C. auriculata leaves when compared with that of their raw leaves. However, boiling has increased the lipoxygenase inhibition ability in S. grandiflora as compared with its steamed leaves, though it is lower than its fresh leave. Interestingly, cooked leaves of G. lactiferum have shown significantly lower (p < 0.05) lipoxygenase inhibition ability when compared with that of its raw leaves.The mechanism of anti-inflammation involves a series of events in which metabolism of Arachidonic acid plays an important role  Arachidohibition . TherefoPlant extracts have been reported to inhibit protein denaturation. Although, the precise mechanism of this membrane stabilization was yet to be elucidated, but it has been proposed that the extract might inhibit the release of the lysosomal content of neutrophils at the site of inflammation . These nP. edulis. C. asiatica, and G. lactiferum showed significantly lower (p < 0.05) inhibition % when compared with that of their raw, boiled, and steamed leaves. Raw leaves of G. lactiferum and S. grandiflora have shown higher proteinase inhibition ability compared with their all cooked forms. However, all of the cooked leaves of C. auriculata have shown similar or higher proteinase inhibition ability compared with that of its raw leaves. For examples, boiling and steaming of leaves of C. auriculata, increased the proteinase inhibitory activity by 54.2% and 56.7%, respectively. Boiled leaves of O. zeylanica exhibited a similar ability of proteinase inhibition to its raw leaves. However, fried and steamed leaves of O. zeylanica showed significantly lower (p < 0.05) proteinase inhibition ability when compared with that of boiled and raw leaves. About 80% reduction in proteinse inhibitory activity was observed in Gymnema lactiferum. According to the In this study, it was found that boiling and steaming process might increase or decrease the anti-inflammatory activities. The frying process reduces the anti-inflammatory activity of all leafy types. The changes in anti-inflammatory activities of these studied leafy types in the cooking process may be due to changes in bioactives, such as polyphenols, flavonoids, and carotenoids. Wachtel-Galor and co-authors  have repCassia auriculata), mella (Olax Zeylanica), Gotukola (Centella asiatica), Ceylon cow tree (Gymnema lactiferum), Kathurumurunga (Sesbania grandiflora), and Passion fruit (Passiflora edulis) were collected in Gampaha and Kurunegala districts in Sri Lanka. All of the chemicals were of analytical grade and were purchased from Sigma Aldrich, St. Louis, MO, USA through Analytical Instrument Pvt Ltd., Colombo, Sri Lanka.Fresh green leafy vegetable samples; Ranawara  were divided into four parts (100 g each), keeping one portion as control , and the rest was subjected to different cooking treatments, as indicated below. Briefly, for boiling, leaf samples (100 g) were added to boiling tap water (150 mL) in a covered stainless-steel pot and were cooked on a moderate flame for 5 min, and then samples were drained off and cooled rapidly on plenty of ice. For steaming, leaf samples were placed on a perforated tray in a stainless steel steamer covered over boiling water for 5 min, and then samples were rapidly cooled on ice. For frying, leaves were added to 500 mL of white coconut oil  in a stainless steel pan at 170 °C and stirred for three minutes until the sample became crisp-tender. At the end of each trial, the samples were drained off and dabbed with blotting paper to allow for the absorption of exceeding oil. The cooked leaf samples were homogenized and stored at −18 °C. As anti-inflammatory activities were calculated according to dry matter basis, moisture contents of the cooked samples were determined according to the method described by Turkmen et al. [Methanolic extracts of leaves were prepared according to the method described by Gunathilake & Ranaweera . Brieflyv/v) suspension with isotonic buffer solution  was NaH2PO4 (0.2), Na2HPO4 (1.15) and NaCl (9.0). Heat-induced hemolysis was carried out, as described by Okoli et al. [Erythrocytes suspension was prepared by the method described by Shinde et al. , with soi et al. , with soThe test was performed following the method described by Gambhire et al. , with sow/v) casein was added. The mixture was incubated further for an additional 20 min. About 2 mL of 70% perchloric acid was added to terminate the reaction. Cloudy suspension was centrifuged, and the absorbance of the supernatant was read at 210 nm against buffer as blank. The percentage of inhibition of proteinase activity was calculated.The test was performed according to the modified method of Sakat et al. , with soPhosphate buffer solution without sample was used as the control. The percentage inhibition of protein denaturation was calculated by using the following Equation (3):Lipoxygenase was assayed, according to the method described by Wu , with soAnalysis of polyphenols was measured, as described in Gunathilake and Rupasinghe , flavonop < 0.05), multiple mean comparisons were carried out using LSD method. Pearson’s correlation coefficients (r), with the level of significance (P ≤ 0.05) (2-tailed) for total polyphenols, flavonoids, and carotenoids versus studied anti-inflammatory results were estimated using MINITAB 15 software. Polyphenols, flavonoids, and carotenoids content of the extracts that were used for correlation studies are based on the same study; however, data are not shown.All data are presented as the mean ± standard deviation for all in vitro assays done. All of the samples were analyzed in triplicate, and one-way analysis of variance (ANOVA) was performed using MINITAB 15 software . When there were significant differences (O. zeylanica and P. edulis. Boiling of leaves increased the inhibitory activity of protein denaturation in C. asiatica and P. edulis; hemolysis in C. asiatica, C. auriculata, and S. grandiflora; lipoxygenase inhibition ability in O. zeylanica, P. edulis, C. asiatica and C. auriculata leaves; proteinase inhibition in C. auriculata when compared with that of raw and their other cooked leaves. The results of the study can be used as a database, providing information on the effects of different cooking methods on the health promotion potential of green leafy vegetables studied.The present study clearly indicates that the in vitro anti-inflammatory associated biological activities of studied green leafy vegetables are modified, increased or decreased, by boiling, steaming, and frying process, depending upon the vegetable species. Among the cooking methods, the frying of all leafy vegetables has reduced the inhibition abilities of protein denaturation, hemolysis, proteinase, and lipoxygenase activities when compared with other cooking methods that were studied. Steaming significantly increased the protein denaturation and hemolysis inhibition in


Congenital nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine. Congenital NDI is mainly caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R), leading to impaired aquaporin-2 (AQP2) water channel activity. So far, treatment options of congenital NDI either by rescuing mutant V2R with chemical chaperones or by elevating cyclic adenosine monophosphate (cAMP) levels have failed to yield effective therapies. Here we show that inhibition of A-kinase anchoring proteins (AKAPs) binding to PKA increases PKA activity and activates AQP2 channels in cortical collecting duct cells. In vivo, the low molecular weight compound 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives increase AQP2 activity to the same extent as vasopressin, and increase urine osmolality in the context of V2R inhibition. We therefore suggest that FMP-API-1 may constitute a promising lead compound for the treatment of congenital NDI caused by V2R mutations. Patients suffering from congenital nephrogenic diabetes insipidus (NDI) fail to concentrate urine due to mutations in vasopressin type 2 receptor (V2R). Here Ando et al. show that agents disrupting the interaction between PKA and AKAPs restore aquaporin-2 activity downstream of V2R, offering a therapeutic approach for the treatment of NDI. Mental retardation, resulting from recurrent severe hyperosmotic dehydration and rapid over-rehydration, is critically related to prognosis3. To avoid these complications, drug discovery for congenital NDI is highly desirable.Congenital nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine. Daytime polyuria with nocturia and polydipsia significantly reduces the quality of life and restricts social activities of patients. In addition, persistent polyuria causes megacystis, hydroureter, hydronephrosis and other urinary tract abnormalities, leading to kidney failure4. To date, serine 256 (S256), 261 (S261), and 269 (S269) in the C-terminus of AQP2 have been identified as major phosphorylation sites related to AQP2 trafficking7. Initial mutational analysis of AQP2 phosphorylation sites suggested that S256 was the sole phosphorylation site responsible for apical translocation of AQP2. However, the generation of phospho-specific antibodies revealed that AQP2 phosphorylation at S256 is constitutively high regardless of vasopressin stimulation8. On the other hand, vasopressin increases AQP2 phosphorylation at S269 and decreases AQP2 phosphorylation at S261. These changes in AQP2 phosphorylation status at S261 and S269 have been well-correlated to translocation of AQP2 to the apical plasma membrane10.Vasopressin type 2 receptor (V2R) and vasopressin-regulated water-channel protein aquaporin-2 (AQP2) are well-established determinants of urine concentration. In response to dehydration, the antidiuretic hormone vasopressin is secreted from the posterior pituitary. Circulating vasopressin increases water permeability of the collecting ducts by rapid translocation of AQP2 to the apical membranes, thereby inducing free water reabsorption from urine to the hypertonic interstitium to prevent further water loss. In this process, vasopressin binds to V2R, thereby activating adenylcyclase, which increases intracellular cyclic adenosine monophosphate (cAMP) production. An elevated cAMP concentration then activates cAMP-dependent protein kinase, PKA, which is thought to be involved in the phosphorylation of AQP211. Most V2R mutants are misfolded in the endoplasmic reticulum and not transported to the cell membrane12. For the treatment of congenital NDI, proper membrane sorting of V2R by restoring protein conformation or bypassing the defective V2R signaling is necessary to activate AQP2. So far, elevating cAMP levels independent of V2R has been mainly studied as a treatment option of congenital NDI. In particular, G protein-coupled receptors (GPCRs), which increase cAMP production in response to their ligands, have been intensively studied17. Nevertheless, these conventional therapeutic approaches have failed to sufficiently activate AQP2 to increase urine osmolality and no specific pharmacological drugs have yet reached clinical application.Almost 80% of all congenital NDI diagnoses are caused by loss-of-function mutations of V2R4. PKA is a tetramer composed of two regulatory (PKA R) and two catalytic (PKAc) subunits in its inactive form. The PKA R subunits have four isoforms: RIα, RIβ, RIIα, and RIIβ. Binding of cAMP to each PKA R subunit causes dissociation of PKAc from the PKA R subunits and subsequent phosphorylation of the consensus target sequence RRXS/T by the PKAc subunit. The intracellular distribution and substrate specificity of PKA are largely controlled by A-kinase anchoring proteins (AKAPs), which serve as scaffold proteins that tether the PKA R subunits and other signaling enzymes in proximity to their target substrates18. In the renal collecting ducts, AKAPs are involved in AQP2 phosphorylation21. AKAPs and PKA probably coordinate AQP2 regulation in the vasopressin signaling pathway; however, little is known about the potential of these molecules for the treatment of congenital NDI. In this article, we report that AKAPs-PKA disruptors, which dissociate the binding of AKAPs and PKA R subunits, increased PKA activity and contributed to AQP2 phosphorylation, trafficking, and water reabsorption. The low molecular weight compound 3,3′-Diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives especially increased AQP2 activity to the same extent as vasopressin. AKAPs-PKA disruptors are potential novel category of therapeutic drugs for congenital NDI and other PKA-related diseases.We focused on direct activators of PKA as novel therapeutic targets of congenital NDI. PKA is known to participate in the mediation of vasopressin-induced AQP2 phosphorylation23. This peptide competes with AKAPs for PKA RII binding and inhibits interactions between AKAPs and PKA RII. Ht31P is a control peptide with a proline substitution at the two isoleucine residues of Ht31. The role of Ht31 in PKA activity differs among target cell lines. Ht31 has been frequently used to inhibit local PKA activity by displacing PKA from its anchored sites and substrates, while Ht31 has been shown to increase PKA activity in baby hamster kidney cells24.The Ht31 inhibitor peptide is a stearated form of a 24-amino acid peptide derived from a human thyroid AKAP Fig. 22,23. Thcl4), which exhibit endogenous expression of AQP2 and AKAPs  at 1 nM, as a positive control. PKA activity was measured by western blot analysis using a phospho-PKA substrate antibody. As shown in Fig. Mouse principal cells of the kidney cortical collecting duct, clone 4  in isolated tubules of the CCDs of mouse kidneys was further examined using a microperfusion technique. As shown in Fig. Pf, as compared to the control values. The effect of FMP-API-1 was equivalent to that of dDAVP. These data indicated that FMP-API-1 certainly increased AQP2 activity and contributed to transcellular water transport across the tubular epithelial cells.As Ht31-induced AQP2 trafficking Fig. , d, PKA 35 and were housed again in metabolic cages for another 24 h to measure water balance. As shown in Fig. Considering that FMP-API-1 activated AQP2 without elevating cAMP, FMP-API-1 appears to be a promising therapeutic target for congenital NDI caused by V2R mutations. Therefore, the therapeutic effects of FMP-API-1 in a V2R-inhibited NDI mouse model were examined. At first, C57BL/6 mice were housed individually in metabolic cages for 24 h to measure basal urine osmolality, urine output, and water intake. The C57BL/6 mice were then subcutaneously infused with tolvaptan or tolvaptan plus FMP-API-1 using osmotic minipumps2), groups that can inhibit the binding of AKAPs and PKA R subunits more efficiently than FMP-API-134, and have more potential to activate AQP2. In the mpkCCD cells, FMP-API-1/27 indeed activated PKA and AQP2 phosphorylation at lower concentrations, as compared with FMP-API-1  were quite different. RNA-seq database analysis revealed that Akap11(AKAP220), Akap12, Cbfa2t3, and Prkar2b (PKA RIIβ) were mostly expressed by the MCD, but not the CCD40. AKAP220 actually colocalizes with AQP2 in the MCD, where it is involved in AQP2 phosphorylation21. These different gene expression profiles between the CCD and MCD cells may explain the more potent effect of FMP-API-1/27 in the mouse kidney, as compared with its effect in mpkCCD cells, a CCD cell line. Moreover, these different expression patterns of AKAPs and PKA in each tissue may contribute to the target-selective property of FMP-API-1/27. The development of other AKAPs-PKA disruptors targeting novel combinations of AKAPs and PKA R subunits is a potential therapeutic strategy for various PKA-related diseases. However, the question of whether AKAPs-PKA disruptors could be used in clinical settings is still controversial, and remains to be addressed. In particular, the precise mechanisms underlying the efficacy of such small molecule inhibitors, including FMP-API-1/27, still need to be elucidated. In addition, FMP-API-1/27 showed little effect in mice by oral administration , suggesting that further modification of the compound would be necessary to improve its pharmacokinetics.FMP-API-1/27 highly activated AQP2 phosphorylation in vivo, rather than in vitro Fig. , c, whic5 because vasopressin increases cAMP/PKA activity and AQP2-S256 is within the PKA targeting motif (RRXS/T). In vitro kinase analysis supports this notion. Purified PKA significantly phosphorylated AQP2 peptide at S2567. Conversely, PKA failed to regulate the AQP2 peptide at S261 and S26941. These results suggest that vasopressin-induced PKA activation selectively increases AQP2 phosphorylation at S256. However, in the mouse kidney, vasopressin regulates AQP2 phosphorylation at S261 and S269, rather than S2568, suggesting that unidentified kinases or phosphatases, other than PKA, are present, in order to account for the detailed mechanisms of AQP2 phosphorylation in the vasopressin signaling pathway42. Importantly, the direct PKA activator FMP-API-1/27 robustly increased AQP2 phosphorylation at S256 in the mouse kidney, which has never been observed before  were cultured as previously described25. The mpkCCD cells were seeded on semipermeable filters . For immunofluorescence and western blot analysis, 0.33-cm2 and 4.67-cm2 filters were used, respectively. The mpkCCD cells were cultured for 5 days, with changes of the medium daily. St-Ht31 , St-Ht31P (Promega Corporation), FMP-API-1 , FMP-API-1/27, FMP-API-1/28, dDAVP (Sigma–Aldrich Corporation), and H89 (Sigma–Aldrich Corporation) were applied to the basolateral side of the mpkCCD cells.mpkCCD35. Urine osmolality was measured with a Fiske One-ten Osmometer . The protocols of the animal experiments were approved by the Animal Care and Use Committee of Tokyo Medical and Dental University.The animal studies were performed using 10-week-old male C57BL/6 mice that were housed individually in metabolic cages with free access to food and water to measure urine osmolality, urine output, and water intake. The mice were subcutaneously infused with tolvaptan  or tolvaptan plus FMP-API-1 using osmotic minipumps  as previously described32. For analysis of the cell fractions, the mpkCCD cells were incubated with 650 µL of 1% Triton X-100 in PBS for 3 min before cell lysis, yielding a detergent-soluble cytosol fraction and an insoluble membrane fraction29. After centrifugation at 15,000 × g for 10 min at 4 °C, the protein concentration was measured using the Bradford protein assay method . The supernatants were denatured in sodium dodecyl sulfate (SDS) sample buffer  for 30 min at 37 °C. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes . The blots were probed with the following primary antibodies: goat anti-AQP2 , rabbit anti-AQP2 , rabbit anti-AQP2 , rabbit anti-AQP2 , mouse anti-β-actin , rabbit anti-phospho-PKA substrate , mouse anti-PKA RIIα , mouse anti-PKA RIIβ , mouse anti-glyceraldehyde 3-phosphate dehydrogenase , and mouse anti-E-cadherin . Alkaline phosphatase-conjugated anti-IgG antibody (Promega Corporation) was used as the secondary antibody and Western Blue (Promega Corporation) was used to detect the signals. The band intensities of the western blots were quantified using ImageJ software (https://imagej.nih.gov/ij/). Uncropped western blots are shown in Supplementary Figure The mpkCCD cells were washed twice with phosphate-buffered saline (PBS) and then solubilized in 200 µL of lysis buffer as previously describedThe mpkCCD cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X/PBS. The filters were detached from the holders and incubated with goat anti-AQP2 antibody . Alexa 488 dye-labeled antibodies  were used as the secondary antibodies. The samples were mounted with Vectashield/4′,6-diamidino-2-phenylindole . Immunofluorescence images were obtained using the Zeiss LSM 510 Meta Confocal Microscope . The fluorescence intensities of AQP2 in the mpkCCD cells were quantified using ImageJ software.Total RNA from the mpkCCD cells and C57BL/6 mouse was extracted using TRIzol Reagent  and purified with RNase-free DNase Sets and RNeasy Kits . RNAs were reverse-transcribed using Omniscript Reverse Transcriptase (Qiagen). Primer sequences used for PCR were summarized in Supplementary Table Prkar2a and Prkar2b coding regions were amplified from mpkCCD-derived cDNA by PCR and were cloned into pENTRegfp2  using the Gibson assembly technique . C-terminal GFP-tagged PKA RIIα and PKA RIIβ were subsequently transferred into pLenti CMV Puro DEST (Addgene #17452) using the LR reaction. The recombinant lentiviral vectors, packaging vector psPAX2 (Addgene #12260) and enveloping vector pMD2.G (Addgene #12259) were co-transfected into HEK 293T cells. The culture medium was harvested and centrifuged at 500 × g for 5 min at 4 °C to remove any cellular debris. The supernatant containing 4 μg/ml polybrene (Sigma–Aldrich Corporation) was added to the mpkCCD cells. The cells were centrifuged at 1,000 × g for 90 min at room temperature to increase transduction efficiency. Successful transduction was confirmed by GFP positive cells under a fluorescence microscope. For molecular analysis mixed populations of cells without antibiotic selection were used. All experiments were evaluated below four passages after transduction. Lentiviral transduction into mpkCCD cells was performed repeatedly to obtain experimental data.The mouse 43.The amount of AQP2 in the apical plasma membrane was quantitated by apical surface biotinylation as previously described44. dDAVP was used as the positive control. Phosphodiesterase inhibitors were not added to the mpkCCD cells.Intracellular cAMP levels were measured with the BioTrak EIA system  as previously described45. The CCD tubules were dissected in cooled (10 °C) dissection solution, then transferred to a 1.5-mL bathing chamber and microperfused for 1 h before measurements of Pf. The perfusion and bathing solutions were bubbled with 95% O2/5% CO2 at room temperature. The perfusion solution contained a volume marker, 0.5 mM fluorescein isothiocyanate-dextran  as previously described46. The bathing solution was exchanged every 30 min to maintain osmolality. The perfusion rates were 5–10 nL/min. The tubule lengths were 427 ± 55 µm (n = 5). Pf was calculated as previously described45.CCD tubules dissected from 10-week-old female C57BL/6 mice were microperfused in vitro. The dissection, perfusion, and bathing solutions were prepared as previously describedn-hexane to yield 3,4-dimethoxybenzophenone as colorless solid . The parameters of proton nuclear magnetic resonance (1H NMR)  were d 7.77 , 7.59 , 7.51 , 7.49 , 7.39 , 6.91 , 3.98 , and 3.96 .Aluminum chloride  was added to a solution of 1,2-dimethoxybenzene  and benzoyl chloride  in methylene chloride at 0 °C, stirred at room temperature for 1 h, then poured into ice-cooled concentrated hydrochrolic acid and extracted with methylene chloride. The organic layer was dried over sodium sulfate and concentrated. The residue was washed with n-hexane/ethyl acetate = 2/1) to yield 4-benzyl-1,2-dimethoxybenzene as colorless oil . The 1H NMR parameters  were d 7.77 , 7.23–7.19 , 6.82 , 6.75 , 6.73 , 4.06 , 3.87 , and 3.84 . Then, 2.0 mL of 1.0 M boron tribromide in methylene chloride was added dropwise to a solution of 4-benzyl-1,2-dimethoxybenzene  in methylene chloride at 0 °C. After stirring at room temperature for 2 h, the reaction mixture was poured into ice-cooled water, and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to yield 4-benzylchatechol as pale yellow solid . The 1H NMR parameters  were d 7.29 , 7.22–7.17 , 6.79 , 6.69 , 6.65 , 5.01 , 4.94 , and 3.88 , while those of 13C NMR  were d 143.6, 141.8, 141.4, 134.6, 129.0, 128.6, 126.2, 121.6, 116.2, 115.5, and 41.3.A solution of 3,4-dimethoxybenzophenone  and triethylsilane  and trifluoroacetic acid (2 mL) was stirred at room temperature for 17 h, then poured into ice-cooled saturated sodium bisulfate and extracted with methylene chloride. The organic layer was dried over sodium sulfate and concentrated. The residue was purified by silica gel column chromatography . The 1H NMR parameters  were d 7.44 , 7.39 , 6.92 , 3.98 , and 3.95 . Then, 1.5 mL of 1.0 M boron tribromide in methylene chloride was added dropwise to the solution of 3,4,3′,4′-tetramethoxybenzophenone  in methylene chloride at 0 °C. After stirring at room temperature for 3 h, the reaction mixture was poured into ice-cooled water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: CHCl3/MeOH = 15/1) to yield 3,4,3′,4′-tetrahydroxybenzophenone as colorless solid . The 1H NMR parameters  were d 9.65 , d 9.39 , 7.16 , 7.05 , 6.82 , while those of 13C NMR  were d 193.2, 149.7, 144.9, 129.3, 122.7, 116.9, and 114.9.A mixture of 3,4-dimethoxybenzoic acid  and 1,2-dimethoxybenzene  in 5 g of polyphosphoric acid was stirred by a mechanical stirrer at 80 °C for 2 ht-test was performed to assess the statistical significance of microperfusion analysis. Data are presented as the mean ± standard error (SE). A probability (p) value of <0.05 was considered statistically significant.Statistical significance was evaluated by a one-way analysis of variance test with multiple comparisons using Tukey’s correction. Data are presented as the mean ± standard deviation (SD). The student’s The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


Circadian rhythms are intrinsic ~24 hour cycles that regulate diverse aspects of physiology, and in turn are regulated by interactions with the external environment. Casein kinase 1 delta  is a key regulator of the clock, phosphorylating both stabilizing and destabilizing sites on the PER2 protein, in a mechanism known as the phosphoswitch. CK1δ can itself be regulated by phosphorylation on its regulatory domain, but the specific sites involved, and the role this plays in control of circadian rhythms as well as other CK1-dependent processes is not well understood. Using a sensitized PER2::LUC reporter assay, we identified a specific phosphorylation site, T347, on CK1δ, that regulates CK1δ activity towards PER2. A mutant CK1δ T347A was more active in promoting PER2 degradation. This CK1δ regulatory site is phosphorylated in cells in trans by dinaciclib- and staurosporine-sensitive kinases, consistent with their potential regulation by cyclin dependent and other proline-directed kinases. The regulation of CK1δ by site-specific phosphorylation via the cell cycle and other signaling pathways provides a mechanism to couple external stimuli to regulation of CK1δ-dependent pathways including the circadian clock. Circadian rhythms are intrinsic ~24 hour cycles of behavioral, neural, hormonal and biochemical processes occurring in most organisms exposed to daily changes in light and dark. These rhythms are controlled by a master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus, which in turn can be reset by external light cues via input from ganglion cells in the retina. The master clock synchronizes intrinsic clocks present in virtually all cells and tissues throughout the body and can couple to the cell cycle in various tissues ,2. CircaPeriod (PER) and Cryptochrome (CRY). PER and CRY proteins are in turn repressors of Clock/BMAL1 activity. PER and CRY proteins accumulate throughout the prospective day, peaking in the early night to turn off their own transcription. PER1 and PER2 undergo multisite phosphorylation by Casein kinase 1 δ and ε (CK1δ and CK1ε) and are degraded, resulting in a trough in protein level in the early day[The vertebrate circadian clock has at its core coupled transcriptional-translational-degradation feedback loops that have been extensively studied and reviewed –10. Clocearly day–14. The The biochemical role of casein kinases 1 δ and ε (CK1δ and ε) in regulating PER stability is becoming clearer. Phosphorylation of PER1 and PER2 at amino acids 478 on mPER2 by CK1δ/ε creates a recognition site for β-TrCP binding ,15. β-Trin vivo phosphorylation sites in the CK1δ and CK1ε carboxyl-terminal tail result from intra-molecular autophosphorylation [in vivo hyperphosphorylation of CK1δ and CK1ε can be induced by short exposure to phosphatase inhibitors such as calyculin A [in vitro assays [Because of its key role in PER phosphorylation and the circadian clock, the regulation of CK1 is of great interest. CK1δ and ε are rapidly and reversibly heavily phosphorylated in vivo and this phosphorylation regulates the activity of the kinases –22. A nurylation . Inhibitrylation –25. Thesyculin A ,21,26. Ro assays ,21,27,28o assays ,29,30. Hin vivo [CK1δ is the key regulator of circadian rhythms in vivo ,32. We hpCK1δ plasmids were pCS2-6Myc-CK1δ (V2418). PF670462, PF4800567 and staurosporine were purchased from Tocris Bioscience. Dephosphorylated casein was purchased from Sigma Aldrich. Cell lines were from American Type Culture Collection (ATCC), USA.Commercial antibodies were sourced as follows: firefly luciferase Ab Abcam ab21176), Myc mAb , CK1δ mAb , β-actin Ab , β-tubulin Ab . Phospho-S478 PER2 [76, Myc m2, unless otherwise stated. For transfection, cells were seeded 1 day pre-transfection to achieve 70% confluency on day of transfection. Cells were transfected with TurboFect (Fermentas) with the indicated amounts of DNA according to manufacturer’s manual.Cells were cultured in DMEM  in the presence of 10% FBS (Gibco), 1x Pen/Strep (Gibco) and 1x Sodium Pyruvate (Gibco), in a humidified incubator conditions at 37°C with 5% COPER2::LUC expression plasmids were transiently transfected alone or with CK1δ/ε in 35mm dishes, in phenol red-free DMEM, in the presence of 0.1 M D-luciferin, 10 mM Hepes and 1.2 g/L sodium bicarbonate. Dishes were sealed with 40 mm cover glasses and vacuum grease, and incubated in the LumiCycle. The next day, 40 μg/ml cycloheximide (Sigma) was added per dish. Luminescence data were used to calculate PER2 half-life in Graphpad Prism using one-phase decay algorithm as described previously. PER2 haCells were washed once in 1x PBS and lysed in either 4% SDS or Hepes Lysis Buffer . SDS lysates were boiled for 5 minutes before being mechanically sheared by syringe and 27½G needle. NP-40 lysates were centrifuged at 13,000 rpm, 4°C for 10 minutes to remove cell debris. Protein quantification was performed using BCA quantification (Pierce #23227). 20–50 μg lysates were added to 4x sample buffer and resolved on SDS-PAGE. Protein ladders used were Bio-Rad’s All Blue (#161–0373) or WesternC (#161–0376) Standards.SDS-PAGE gels were 10% unless otherwise stated. Resolved lysates were transferred to PVDF membranes (Immobilon IPFL00010) in a wet transfer system in transfer buffer . Membranes were then blocked in 5% BSA. Depending on the antibodies, visualization was performed on either by fluorescence on Licor-Odyssey, or by enhanced chemiluminescence on ImageQuant  using Immunestar or Clarity (Bio-Rad 170–5061) ECL reagent.in vivo phosphorylation sites on CK1δ/ε were identified from the PhosphoSite Plus database on 1 December, 2015 (www.phosphosite.org). cDNA sequences of mutant carboxyl-terminal tails were synthesized by GeneScript in a pUC57 backbone, and used to replace CK1δ wildtype sequence by EcoRV blunt ligation. cDNA were PCR amplified with primers N1073 (CCGGCAGGGCTTCTCA|TATGACTACGTGTTCGACTG) and N1075 (CATTCCACAGGGTCGA|CCACTGTGCTGGCGAATT) using Platinum Taq kit. pBABE-6Myc-CK1δ (V1250) was engineered to carry an internal NdeI cut site using site-directed mutagenesis. These were then linearized using NdeI and SalI. Linearized backbone and PCR products were resolved on agarose gel electrophoresis, excised, and purified using QiaQuick gel extraction kit. PCR products were then cloned into linearized backbones using ColdFusion cloning kit (SBI MC101B-1), at a 2:1 insert:vector molar ratio.Putative Constructs for transient transfections were made by cloning from the pBABE constructs into pCS2 vector. The pBABE constructs were digested with ClaI and AvrII. pCS2-EV (V513) was digested with ClaI and XbaI. Digested products were resolved on agarose gel electrophoresis. Bands of interest were then excised and purified using MN NucleoSpin Extract II kit. Ligation of vector and insert was performed using Fermentas Rapid Ligation Kit.Cells were lysed in kinase assay lysis buffer (50 mM Hepes (pH 7.5), 1% NP-40, 0.1% SDS, 150 mM NaCl, 20 mM β-glycerol phosphate, 1mM DTT and 1x protease inhibitor cocktail), and centrifuged 13, 000 rpm, 4°C, 15 minutes. Immunoprecipitation was performed using the stated antibody with overnight tumbling at 4°C. Protein A/G beads slurry was added the next morning, and tumbled a further 2 hours at 4°C. Beads were pelleted at 1,500 rpm for 5 minutes. Beads were then washed once with kinase assay lysis buffer, once with kinase assay lysis buffer without SDS and DTT, and twice with CK1 storage buffer , 15 mM NaCl, 1 mM DTT). Beads were split into 3 fractions at the final wash step before buffer removal.2, 0.5 mM DTT, 150 mM KCl, 10 μg dephosphorylated casein (Sigma), 40 nM Calyculin A) was added to one set of beads and incubated on ice for 5 minutes. ATP mix (2 μCi 32P-ATP (Perkin Elmer #BLU502A250UC), 100 μM ATP) was then added to the beads and incubated at 37°C for 15 minutes. To pre-autophosphorylate, ATP mix and reaction buffer with casein omitted was added to a second set of beads and pre-incubated at 37°C for 15 minutes, before the addition of casein followed by an additional incubation at 37°C for 15 minutes. All reactions were stopped by the addition of 6x SDS-sample buffer. The samples are resolved on 4–20% gradient SDS-PAGE (Biorad), the gel stained with Coomassie Blue (Expedion Instant Blue ISB1L), and exposed to a Phosphor Screen overnight. Screen is imaged on the Typhoon FLA9500 . One set of beads were used for western blot to check for equal immunoprecipitation and loading.Substrate mixture (20 mM Tris-Cl (pH 8.0), 5 mM MgClPrimers were designed using Stratagene’s primer design software. 20 ng template DNA was used for site-directed mutagenesis with Pfu Ultra (Stratagene), according to Quikchange protocol. Methylated DNA was digested with DpnI (NEB) at 37°C for 2 hours. 2 μl of the reaction mix was transformed into Top10 cells. Plasmids were extracted using Machery-Nagel miniprep kit.Cells were washed once with 1x PBS, and lysed in HTS buffer ). Lysates were centrifuged at 13,000 rpm at 4°C, for 10 minutes to remove cell debris. Supernatants were collected and pre-cleared with 10 μl of Protein A/G beads slurry with tumbling for 30 minutes at 4°C. Protein quantification was performed using Bradford’s assay (Bio-Rad #500–0002). 500 μg protein were immunoprecipitated with 1 μg antibodies indicated, unless otherwise stated. Immune-complexes were allowed to tumble overnight at 4°C. Following morning, 20 μl agarose Protein A/G were added. Immune-complexes were allowed to tumble at 4°C for 2 hour. Beads were washed 3 times with 1 ml lysis buffer without DTT and protease inhibitor. Elution was performed using 2x sample buffer and boiled at 95°C for 5 min.All statistical analyses were done using Graphpad Prism. Half-lives were calculated using the one-phase decay algorithm using the luciferase activity, from the point of cycloheximide addition to the plateau at minimum luciferase activity. All half-life calculations shown are of three independent experiments done in duplicates. Adjusted p-values were calculated using 1- or 2-way ANOVA with Tukey’s post-hoc correction using GraphPad Prism. In all figures, * indicates p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.in vivo [There are at least two major CK1 phosphorylation sites on PER2 with opposing consequences for PER2 stability. Phosphorylation in the FASP region stabilizes the protein, while phosphorylation at the β-TrCP site destabilizes PER2 ,13,17–19in vivo . We tookWe have previously shown that mutation of mouse PER2 S659 increased the phosphorylation of PER2 at S478 by CK1ε . To confTo test if S478 was the major phosphorylation site driving PER2 degradation, wildtype, S659A or S478A/S659A double mutant PER2::LUC were co-expressed with increasing amounts of CK1δ. Mutation at S478 partially restored PER2(S659A) stability . This suTaken together, these data demonstrate that PER2(S659A)::LUC provides a sensitized background to investigate the effects of changes in CK1 activity, mediated, at least partially, through increased phosphorylation of PER2 at the destabilizing S478 site.Per2(S659A)-luc plasmid  than that seen in cells receiving 50 ng plasmid (6.4 hrs). Importantly, increasing amounts of CK1δ expression decreased PER2::LUC half-life in a dose dependent manner, with half-maximal decrease occurring at ~500 pg CK1δ expression plasmid (To more quantitatively assess the effect of CK1δ activity on PER2(S659A) degradation, picogram and nanogram quantities of CK1δ expression plasmid were co-transfected with only 10 ng of  plasmid . This ambroblasts,33. Nota plasmid . ConsistTo determine if this increase in PER2 degradation was kinase activity dependent, we co-expressed catalytically inactive CK1δ K38A with PER2(S659A)::LUC. Unlike CK1δ, CK1δ(K38A) expression had no effect on PER2 half-life until the highest levels, when the half-life increased, consistent with a dominant negative effect of CK1δ(K38A) .in vitro autophosphorylation of bacterially expressed recombinant protein [in vivo phosphorylation sites on the CK1δ carboxyl terminal tail were identified using PhosphoSite Plus [in vivo is actively reversed by serine-threonine phosphatases that can be inhibited by phosphatase inhibitors [CK1δ can be phosphorylated on its carboxyl terminal tail and this is inhibitory to its activity ,22. Earl protein . In the ite Plus . This aphibitors ,21. Whil32P-ATP incorporation was seen both in CK1δ itself due to auto-phosphorylation, as well as in casein ::LUC, and PER2 stability was measured. We noticed that the  WT Figs  and 4A,  WT Figs . When pr WT Figs .To determine the specific phosphorylation site(s) on CK1δ responsible for the increased kinase activity, we screened a series of individual phosphorylation site mutants, based on the information at Phosphosite. We found that T347A mutation could phenocopy the effect of the CK1δ NP mutant . Given CIn order to study T347 phosphorylation, we generated a phospho-specific antibody against CK1δ pT347. Confirming the specificity of the phospho-epitope specific antibody, when CK1δ was overexpressed in the presence of Calyculin A, the antibody detected phospho-CK1δ in wild-type but not T347A CK1δ . CalyculBased on the finding that autophosphorylation resulted in inhibition of CK1δ kinase activity, we initially presumed that the inhibitor phosphorylation sites in the CK1δ tail were the result of autophosphorylation. To test that assumption, kinase-dead CK1δ was transiently overexpressed. Unexpectedly, catalytically inactive CK1δ K38A was still phosphorylated on T347 in the presence of Calyculin A . The K38Our data on the role of phosphoT347 indicates changes in the phosphorylation of this site regulate circadian rhythms. Inspection of the sequence context of T344 and T347  suggestePeriod protein degradation in the accumulation phase of the circadian cycle is regulated by CK1δ/ε. The critical importance of CK1 activity in circadian rhythms is highlighted by the number of sporadic mutations in mammalian CK1δ, CK1ε, and CK1 phosphorylation sites that alter period ,37–39. CEuglena gracilis showed synchronization of cell division by illumination [The effect of proline directed kinases such as the cyclin-dependent kinases on CK1δ activity and hence on PER2 stability suggests a mechanism for cell cycle signaling events to influence circadian period, as has been recently suggested . The conmination . It was mination . Our resmination . The potSeveral external events can regulate circadian clocks and could function through changes in CK1 activity. Signals from the retinohypothalamic tract impinge on SCN and other neurons to alter period in response to changing light conditions. It is relevant here that metabotropic glutamate receptor signaling can regulate CK1 activity via phosphorylation. Cells iIn summary, we describe here a specific mechanism for the regulation of CK1δ activity by proline-directed kinases, and demonstrate the relevance of this regulation in the control of a key CK1δ substrate, the PER2 protein. Given the importance of CK1δ and CK1ε in diverse signaling pathways, these findings in addition have a broader impact on our understanding of how these essential regulators are regulated.


Although aberrant visual attention has been identified in infants at high familial risk for autism, the developmental emergence of atypical attention remains unclear. Integrating biological measures of attention into prospective high-risk infant studies may inform more nuanced developmental trajectories, clarifying the onset and course of atypical attention and potentially advancing early screening or treatment protocols. Heart rate-defined sustained attention (HRDSA) is a well-validated biological measure of attentional engagement that, in non-clinical infant populations, provides incremental information about attentional engagement beyond looking behaviors alone. The present study aimed to examine the characteristics and clinical correlates of HRDSA in high-risk infants, informing whether HRDSA may operate as a promising biological measure of attention and clinical symptoms in this population.n = 21) compared to low-risk controls (n = 21), with most participants contributing multiple assessments. Emergent autism features were measured using the Autism Diagnostic Observation Schedule at 24 months. Primary dependent variables included the proportion of time in behavioral attention, proportion of time in HRDSA, and average heart rate deceleration during HRDSA. For each variable, we used nested multilevel models to examine whether attention differed by group, as well as whether attention predicted emergent autism features among high-risk infant siblings.We examined age-related patterns of HRDSA during a passive looking task in 5- to 14-month-old high-risk infant siblings of children with autism (As expected, HRDSA served as a global biological measure of attention in high-risk infants, predicting greater variability in group risk status than behavioral looking alone. Among high-risk infants, more severe ASD features were also associated with increasingly shallow heart rate deceleration during HRDSA across development, suggesting abnormal qualities of HRDSA may inform individual differences within this population.These preliminary findings provide initial evidence that HRDSA may offer a sensitive, affordable, and portable biological measure of attention that may enhance understanding of atypical attention in high-risk infants. Using this method, we also provide initial evidence that atypical patterns of heart activity previously reported in children and adults with autism may emerge in the first year of life, warranting further study of how HRDSA may specifically inform attention profiles in ASD. Visual attention is one of the most robust early indicators of autism spectrum disorder (ASD), with higher rates of atypical features identified prior to age 12 months in infants later diagnosed with ASD . AlthougIt is well-established that infants’ physical look durations do not cleanly map onto attention, as infants will often continue looking when they are no longer cognitively engaged in stimulus processing . Thus foFew studies have examined heart activity in HFR infants, and it is possible that HRDSA may present differently in HFR given the well-established autonomic abnormalities associated with ASD  and 12-month controls (n = 10), infants with FXS generally exhibited shallower heart rate decelerations and lower heart rate variability during a visual attention task [More recent work has begun expanding studies of HRDSA to infants at risk for ASD. In a longitudinal pilot study of 9- to 18-month infants with FXS . Moion task . Specifiion task ), which ion task ), which Although studies of HFR infants have critically advanced understanding of the emergence and early symptoms of ASD in infants, a major challenge to this work is the vast heterogeneity in individual profiles and features that present in infants later diagnosed with ASD. Biological measures such as HRDSA may improve HFR infant research by supporting more precise measurement of individual differences, reducing error and increasing power in HFR infant attention studies. Indeed, although abnormal cognitive and social attentional features have been observed in HFR infants and have been the topic of several reviews , 37, attn = 9) who exhibited shorter peak looks on social trials of a habituation task compared to HFR infants who did not meet ASD criteria (n = 49), although group differences were no longer significant at 12 months [Relevant to the present study, a number of studies suggest abnormal attentional patterns in HFR infants across a variety of visual attention tasks, with particularly atypical profiles among infants with later ASD features. For example, HFR infants who later meet ASD diagnostic criteria exhibit longer saccadic latencies to disengage attention from competing stimuli , 38 and 2 months . Limited2 months , and oth2 months . Reflect2 months .Improving the quality and depth of attention measurement during visual attention tasks, such as through the integration of HRDSA, may help clarify the scope, emergence, and mechanisms of abnormal attention in this population. At the phenotypic level, examining HRDSA may further inform whether sustained attention is disrupted in HFR infants, particularly those with later ASD features, building on a single study that has explicitly addressed this topic to date . MethodoHeart-rate defined sustained attention is a promising biological measure for measuring and tracking the development of attention in HFR infants given both the popularity of this method in typical developmental literature and its emergent promise in characterizing phenotypes in infants with FXS . To testGiven the exploratory nature of this study, we aimed to test two distinct hypotheses regarding the nature of HRDSA in HFR infants. The first hypothesis was that HFR infants would display greater HRDSA, indexed by greater proportion of time in HRDSA and deeper heart rate decelerations during HRDSA, and that these patterns would be most atypical in infants who later exhibited more severe ASD features. This hypothesis reflects a global assumption that HRDSA would relate to behavioral sustained attention similarly across HFR and LFR groups, with greater HRDSA paralleling greater behavioral attention, similar to previous studies of typically developing infants , 19. Indshallower heart rate decelerations during HRDSA, similar to enhanced behavioral attention and shallower heart rate decelerations previously observed in fragile X syndrome [Our alternative hypothesis was that HFR infants, particularly those with greater emergent ASD features, would display enhanced behavioral attention yet syndrome . This hysyndrome –31, as wsyndrome . In othen = 15), technical difficulties (n = 9), and the infant refusing to sit (n = 1). Groups exhibited similar numbers of participants with one , two , and three assessments .Participants were drawn from an ongoing study of early development in HFR infants. The final sample included 42 infants  assessed between 5 and 14 months of age across 73 observations. Table Procedures were approved by the Institutional Review Board. Parents provided consent prior to the onset of the study and were compensated for participation. To minimize family travel, attention assessments alternated between laboratory and home environments. Experimental setup was standardized across settings using a portable, nonreflective black felt shield. As part of a larger battery, each assessment included a passive viewing attention task and additional developmental and clinical evaluations. The attention task was attempted at the beginning of each assessment and was later re-attempted if the infant was fussy or would not comply. The present study includes behavioral and heart activity data from all assessments, as well as developmental testing conducted around 12 months of age. Follow-up ASD diagnostic assessments were conducted in the laboratory around 24 months.Baby Einstein children’s video depicting animal puppets, moving toys, and classical music. Participants were seated in a darkened room, 10 in. away from an “11 × 24” LCD monitor. Two video cameras simultaneously recorded stimuli and participants’ faces to capture whether infants were looking at the screen. Electrocardiogram signal (ECG) was collected using Alive heart monitors , and signal was transmitted live to a laptop via Bluetooth. If the child did not look at the video voluntarily, the examiner provided up to three prompts to re-engage the child, after which the child was permitted to look toward or away from the screen.During each assessment, participants’ looking behavior and heart activity were measured while they viewed an engaging 135-s The Autism Diagnostic Observation Schedule—Toddler Module (ADOS-T)  is a welThe Mullen Scales of Early Learning (MSEL)  is a staBehavioral data were integrated into Observer XT 10.1 software  for offln = 15; 6 HFR infant, 9 LFR), consistent with previous studies of HRDSA in ASD [Heart activity artifact editing was completed by a coder trained to research reliability through a standardized training sequence supervised by the Brain-Body Center staff . Files tA in ASD . Data weA in ASD . Next, dA in ASD  in SAS 9F = 0.04, p = .85, mean chronological age across assessments, F = 0.62, p = .44, or developmental level at outcome F = 1.50, p = .23. Developmental level did not correlate with any dependent variable across the sample or within either group. However, developmental level was lower among HFR infants who exceeded the ADOS diagnostic threshold ; thus, ADOS models were repeated with developmental level covaried to ensure effects were not driven by lower developmental level. The following variables which were log-transformed due to non-normality: proportion looking time, proportion time in HRDSA, average IBI deceleration during HRDSA, and ADOS CSS.Analyses were conducted using SAS 9.4  with α set to less than 0.05. Data were evaluated for analytic assumptions and outliers prior to analyses. Groups did not differ in number of assessments per participant, Analyses focused on three primary dependent variables: proportion of time in behavioral attention, proportion of time in HRDSA, and average depth of heart rate deceleration during HRDSA. To test for the incremental effects of HRDSA over behavioral looking alone, we controlled for the main effect of behavioral attention (centered at the mean) and the interaction between behavioral attention and the relevant level 2 predictor (group or ADOS CSS) when conducting models examining HRDSA. Due to the nested structure of participant data, we analyzed levels and change in attention using mixed effects multilevel models. Preliminary ICC and model fit analyses indicated that data clustered within individuals; however, the association between dependent variables and age was relatively stable across individuals. Thus, only intercepts were modeled as random effects, and age effects are interpreted as age-related patterns that account for nesting within individuals, rather than differences in individual growth curves.R2 [For our first step of analyses, we characterized general patterns of each variable across age using unconditional models. Next, we constructed a series of conditional models to test the hypotheses that HFR infants would be more behaviorally attentive and display greater HRDSA, indexed by greater proportion of time in HRDSA and deeper heart rate decelerations during HRDSA. These models tested the effect of group membership (level 2 predictor) on initial level and change of behavioral attention (proportion time looking toward screen) and heart rate-defined sustained attention . Finally, we constructed a second set of conditional models to test whether among HFR infants, higher severity of ASD symptoms (ADOS CSS) would be associated with more abnormal behavioral attention and HRDSA. To do so, we repeated the multilevel analyses in the HFR infant group only, replacing group with ADOS CSS as the level 2 predictor. For each conditional model, we estimated proportion of variance explained by predictors using pseudo R2 .Preliminary unconditional models indicated that across the sample, each primary dependent variable  was relatively stable across age. However, as expected, conditional models indicated group differences in behavioral attention, with the HFR group failing to exhibit typical decreases in attention across age. Fixed effects for these models are presented in Table Controlling for behavioral attention, groups differed in the proportion of time spent in HRDSA, with combined predictors accounting for 54% of model variability. Figure Contrary to hypotheses, groups did not differ in average heart rate deceleration during HRDSA. Figure p = .002), suggesting the association between heart rate deceleration and ADOS scores was not driven by lower cognitive abilities in children with higher ADOS scores. Thus, among HFR infants, higher ADOS scores were associated with increasingly blunted heart rate decelerations during HRDSA across age.We next examined the association between ASD features and attention within the HFR group only. Participants’ ADOS CSS were associated with abnormal age-related patterns of heart rate deceleration during HRDSA. As depicted in Fig. Integrating low-cost, accessible biological measures of attention in HFR infants may advance efforts to clarify the attentional phenotype of HFR infants, including potential predictors of later ASD features. Although abnormal attention and heart activity are well-documented in ASD and have been posited to relate to ASD emergence , the intOur first major finding was that proportion of time in HRDSA provided a more sensitive index of group status than behavioral looking alone. This finding generally supports HRDSA as a valid biological measure of sustained attention in HFR infants, paralleling previous studies in which HRDSA converged with behavioral engagement during social attention paradigms in children with ASD , 34. CriNotably, the group differences we observed in sustained attention across HFR and LFR groups—indexed both behaviorally and autonomically—also advance the HFR infant literature. As we expected, HFR infants failed to exhibit typical decreases in behavioral attention and HRDSA observed in LFR controls, manifesting in increased attention among HFR infants at older ages. Behaviorally, these patterns parallel previous studies in which HFR infants, particularly those with later ASD features, display reduced visual engagement at 6 months , fail toqualities of attention engagement that are related to emergent ASD features not detectable by quantities of attention alone. From a theoretical perspective, these findings suggest that dysregulated HRDSA may be relevant to emergent ASD features in HFR infants. Although preliminary, this finding maps onto several relevant studies of heart activity in neurodevelopmental disorders. First, these patterns provide preliminary but novel evidence that autonomic dysfunction observed in children and adults with ASD [Our second major finding was that among HFR infants, later ASD symptoms were uniquely predicted by increasingly shallow HRDSA across age, despite ASD symptoms remaining unrelated to overall time spent in behavioral attention or HRDSA. These data suggest that in addition to providing a valid index of sustained attention, HRDSA may be sensitive to with ASD , 13 may with ASD  and fragwith ASD . Our finwith ASD . Intereswith ASD . Togethen = 9), and our primary focus on ASD features continuously rather than a categorical diagnosis. It will also be important to clarify how methodological issues such as task design  and stimulus types  may affect findings. Further work is also needed to clarify whether global arousal atypicalities and attention-specific impairments present as additive or multiplicative components of risk, as it is unclear whether abnormal HRDSA is a downstream effect of broader autonomic dysregulation reported in ASD. In addition, it will be important to establish long-term profiles and developmental cascades associated with abnormal heart activity in HFR infants, including establishing the specificity of these associations to ASD and the stability of these associations over time. Finally, future work should also examine the association between HRDSA and ASD in across multiple high-risk samples simultaneously. For example, comparing HRDSA in HFR infants and infants with FXS may clarify the specific correlates of ASD that span multiple groups, given ASD features were associated with decreased heart rate decelerations in our HFR sample versus increased decelerations in FXS [This preliminary study supports the need for further work examining HRDSA as a biological measure of attention in HFR infants. However, as with any biological measure relevant to ASD, further study of HRDSA should rigorously characterize the scope and limitations of applying HRDSA to HFR infant work, as well as carefully validate any future translational efforts . It wills in FXS . This wos in FXS  by proviThe present study provides initial evidence that HRDSA may offer a sensitive, affordable, and portable biological measure of attention that may enhance understanding of the intersection of heart activity and attention in HFR infant samples. Using this method, we also provide initial evidence that atypical patterns of heart activity previously reported in children and adults with ASD may emerge in the first year of life among a subset of HFR infants, warranting further study of how HRDSA may specifically inform emergent ASD features. This work warrants further study of HRDSA as a potential biological measure of attention and clinical risk in HFR infants.


A receptors and, interestingly, GABAB receptor mediated components. This study provides the first in-depth characterization of Chrna2 cells in the subiculum, and suggests that subiculum and CA1 Chrna2 cells are generally similar and may play comparable roles in both sub-regions.The nicotinic acetylcholine receptor alpha2 subunit (Chrna2) is a specific marker for oriens lacunosum-moleculare (OLM) interneurons in the dorsal CA1 region of the hippocampus. It was recently shown using a Chrna2-cre mice line that OLM interneurons can modulate entorhinal cortex and CA3 inputs and may therefore have an important role in gating, encoding, and recall of memory. In this study, we have used a combination of electrophysiology and optogenetics using Chrna2-cre mice to determine the role of Chrna2 interneurons in the subiculum area, the main output region of the hippocampus. We aimed to assess the similarities between Chrna2 subiculum and CA1 neurons in terms of the expression of interneuron markers, their membrane properties, and their inhibitory input to pyramidal neurons. We found that subiculum and CA1 dorsal Chrna2 cells similarly expressed the marker somatostatin and had comparable membrane and firing properties. The somas of Chrna2 cells in both regions were found in the deepest layer with axons projecting superficially. However, subiculum Chrna2 cells displayed more extensive projections with dendrites which occupied a significantly larger area than in CA1. The post-synaptic responses elicited by Chrna2 cells in pyramidal cells of both regions revealed comparable inhibitory responses elicited by GABA The hippocampus is a multi-modal structure long known to serve critical functions in spatial information processing, learning, and memory. Interneurons are believed to play a central role in hippocampal function, being at the interface between the hippocampus network which they can powerfully modulate, and inputs to this network . Among tThe subiculum is the major output structure of the hippocampus . It has The subiculum has also been found to acts as an intrinsic generator of hippocampal theta rhythm , oscillaThe goal of this study is to characterize Chrna2 cells in the subiculum compare them to Chrna2 cells in CA1. Using whole-cell patch clamp and optogenetic techniques, we characterized subiculum Chrna2 cells and the post-synaptic responses they elicit in pyramidal cells. These properties were compared to those of CA1 Chrna2 cells to determine whether subiculum Chrna2 cells are a distinct population. We found that subiculum and CA1 cells were similar, with respect to their Som expression and electrophysiological properties. While subiculum Chrna2 cells displayed more extensive dendritic trees and deep layer axonal branching than those in CA1, they evoked similar post-synaptic inhibitory responses in pyramidal cells.ad libitum.This study was carried out in accordance with the recommendations of the Canadian Council on Animal Care and the McGill University Animal Care Committee. The protocol was approved by the McGill University Animal Care Committee. Animals were housed in a temperature-controlled room with a 12/12 h light/dark cycle and food and water Chrna2-cre transgenic C57BL/6 mice . In this recently developed mouse line, cre recombinase is expressed under the Chrna2 promoter (RRID:IMSR_JAX:007905) to generate Chrna2-Tom mice in which tdTomato (Tom) is exclusively expressed in cells expressing cre recombinase. Tom fluorescence was used to identify Chrna2 cells.This study made use of promoter . As descpromoter ; thus, tRRID:AB_2195930) for 16 h at 4°C, followed by an anti-rabbit secondary antibody coupled to Alexa488  for 2 h at room temperature to visualize Som+ cells. Slices were mounted and one image was acquired at 10× magnification in both CA1 and subiculum for each slice using an Axio Observer microscope . Som+ and Tom+ cells were counted using ImageJ software1 (RRID:SCR_003070).Immunohistochemistry experiments were used to determine whether Chrna2 cells in the subiculum express Som. Chrna2-Tom animals were intracardially perfused with paraformaldehyde (PFA) and brains were harvested as described in 3, 10 mM glucose, 3 mM KCl, 2 mM MgCl2, 1.25 mM NaH2PO4, and 1 mM CaCl2, continuously oxygenated with 95% O2/5% CO2; pH 7.3). Horizontal or coronal slices (300 or 400 μm) were cut using a vibratome  and transferred to an artificial cerebrospinal fluid (aCSF) solution at room temperature  for 30 min before recording. Slices were recorded in a bath of aCSF perfused at a rate of 2 ml/min and heated to 30°C . Chrna2 cells were identified by Tom fluorescence using an upright BX51WI Olympus microscope with a 60× immersion objective  and an X-cite Series 120Q fluorescence system . Glass patch pipettes  had a resistance 2.0–6.0 MΩ and were filled with intra-pipette solution . Chrna2 cells were recorded using a visually guided whole-cell patch clamp technique, a MultiClamp 700B amplifier, a DigiData 1440A digitizer and pClamp10 software, and analyzed with Clampfit10 Software . Recordings were kept for analysis only if spikes overshot 0 mV and access resistance was <30 MΩ. There was no correction for junction potential (-9 mV). Membrane resistance (Rm) and access resistance (Ra) were measured in voltage clamp (vc) using pClamp10 software. The following properties were assessed in current clamp (cc). Resting membrane potential (Vr) and spontaneous spiking were assessed over a 30 s recording with no holding current. To assess spike properties, cells were held at a holding potential (h.p.) of -60 mV and a series of 600 ms depolarizing current steps was applied. The step which elicited the first spike was used to assess spike amplitude and half width, and after-hyperpolarization (AHP) amplitude and time. The same series of current steps was applied at an h.p. of -80 mV. The current step which elicited the first spike at -80 mV was noted and the response to a step of equal current applied at -60 mV was analyzed to determine firing rate, peak interspike interval (ISI) (ISI for the first two spikes), steady state ISI (ISI for the last two spikes), and accommodation [(steady state ISI - peak ISI)/steady state ISI]. To assess sag, if present, a series of hyperpolarizing current steps was applied at an h.p. of -60 mV. The step which hyperpolarized the cell to -120 mV was used to calculate sag amplitude, measured as the difference between peak and steady state hyperpolarization, and to determine the presence of a rebound spike. The properties of subiculum and CA1 cells were compared.To assess the electrophysiological properties of CA1 and subiculum Chrna2 cells, patch clamp experiments were performed in Chrna2-Tom animals. Mice were killed by decapitation, and the brain was dissected and placed in an ice-cold high-sucrose solution  to the intra-pipette solution. To obtain efficient biocytin fills, cells were subjected to a high amount of current (700–800 pA for 100 ms) at the end of each recording session  at 4°C overnight. Fixed slices were incubated with streptavidin Alexa 488  in 0.3% PBS-Triton overnight at room temperature. Slices were mounted on glass slides with Fluoromount G .Biocytin-labeled Chrna2 cells were imaged under the MBF Zeiss ApoTome.2 microscope with a 20× magnification . The cells were reconstructed manually using Neurolucida 11 software . Dendrites were distinguished from axons by the presence of spines. Initial axon segments were identified by their smooth appearance near the soma which later appeared discontinuous as the axon continued branching. Due to slicing techniques, neuronal processes may be cleaved, leaving abrupt endings visualized as ball-like structures at the end of the processes. The dendrites and axons for cells were noted for abrupt endings, and all cells that were filled for 30 min and had no visualized swellings in their axonal or dendritic processes were considered for neuron tracing. Area and perimeter occupied by the dendritic tree were assessed using convex hull analysis. Dendritic complexity was assessed using Sholl analysis , in whicRRID:SCR_012730) was injected at the dorsal CA1/subiculum border . Following surgery, animals were returned to their home cage. Experiments were performed 2–4 weeks post-surgery to allow for virus expression.To investigate post-synaptic responses elicited by Chrna2 cell activation in pyramidal cells, an optogenetics approach was employed. This allowed the precise activation of Chrna2 cells. The opsin ChETA, a humanized variant of the excitatory opsin ChR2 [hChR2(E123T/T159C)] with faster on/off kinetics and improved spike fidelity at higher frequency stimulation  was deliRRID:AB_523903), and a rabbit anti-RFP primary antibody, reactive with Tom , followed by an anti-goat secondary antibody coupled to Alexa488  and an anti-rabbit secondary antibody coupled to Alexa568 . Slices were imaged using a Zeiss Imager.M2 microscope with a 10× objective  and Stereo Investigator 13 software (RRID:SCR_002526). The injection site was located, and one slice per 100 μm was analyzed for 500 μm rostral and caudal to this location. One image stack was acquired at 10× magnification in both CA1 and subiculum for each slice, and YFP+ and Tom+ cells were counted using ImageJ software .At post-injection day 22–24, Chrna2-Tom animals were perfused (protocol as described above) for use in immunohistochemistry experiments to validate virus expression. The immunohistochemistry protocol was as described above with the following antibodies: a goat anti-GFP primary antibody, reactive with eYFP  A 1 mm optic fiber connected to a blue LED  was positioned over the hippocampus . To first ensure that Chrna2 cells could be driven by blue light, ChETA-expressing Chrna2 cells, as identified by YFP expression, were patched and responses to blue light pulses were recorded. Photocurrent was measured at the end of a 500 ms light pulse in vc at an h.p. of -70 mV (mean of 10 recordings per cell). Spiking parameters were assessed in cc during light pulses of 1, 5, 10, 20, 50, and 500 ms at an h.p. of -60 mV. Frequency of firing was measured from 500 ms light pulses (mean of 10 recordings per cell). The mean delay between light onset and spike start was measured for the first spike elicited by a 500 ms light pulse (mean of three recordings per cell). To assess spike fidelity, light pulses of 1, 5, and 10 ms were delivered in trains of 1, 2, 4, 6, 8, 10, 20, 40, 60, 80, and 100 Hz .Vr (-56.3 ± 1.2 mV) that was far enough from spike threshold to allow little to no spiking in all cells. Amplitude of the response was examined in vc across different h.p.’s to determine the reversal potential. Paired pulse recordings were also performed in vc for 10 ms light pulses at an h.p. of -60 mV at delays of 50, 100, 200, 500, and 1000 ms. Paired pulse ratio was calculated as amplitude of second/first response from the mean of 10 recordings per condition.To characterize pyramidal cell post-synaptic responses elicited by Chrna2 cell activation, putative pyramidal cells, as identified by shape, position, and firing properties, were patched in subiculum and CA1 during optogenetic stimulation of Chrna2 cells. Regular vs burst firing subiculum pyramidal cells were identified by responses to 600 ms depolarizing current steps. Burst firing pyramidal cells displayed at least one burst during the current step (burst = two or more spikes at a frequency ≥100 Hz). Responses to blue light pulses of 5, 10, 20, and 50 ms were recorded in both vc and cc at h.p.’s from -45 to -80 mV in steps of 5 mV. Analyses were conducted on the mean of 10 recording per condition for each cell. If a spontaneous event, such as a spike, obscured the post-synaptic response, this recording was omitted from the mean. Any condition for which fewer than three recordings remained after these omissions was not included in the analysis. Amplitude, half width, decay time, and delay between light onset and post-synaptic response start or peak were analyzed and compared between pulse widths and pyramidal cell types at an h.p. of -60 mV. This h.p. was chosen as it was the potential closest to mean ARs were blocked using SR95531 . GABABRs were blocked using CGP-55845 hydrochloride . Responses to blue light pulses of 5, 10, 20, and 50 ms were recorded in cc at h.p.’s from -45 to -80 mV  after the addition of gabazine, and again after the subsequent addition of CGP-55845. Amplitude, half width, decay time, and delay between light onset and post-synaptic response start or peak were analyzed at an h.p. of -60 mV and pulse width of 50 ms. This pulse width was chosen as, in all but one cell (n = 14/15), a component of the response remained after gabazine application; a larger proportion than for other pulse widths.To determine which receptors play a role in post-synaptic responses to optogenetic activation of Chrna2 cells, pharmacological methods were employed to block GABA receptors. GABAt-test for paired-pulse ratios, two-tailed paired or unpaired Student’s t-tests for two-group comparisons and two-way ANOVAs with Bonferroni post hoc two-tailed paired Student’s t-tests for multi-factor comparisons. ANOVAs were corrected for sphericity with the Greenhouse–Geisser correction. Data that were not normally distributed were compared with the following non-parametric tests: two-tailed Mann–Whitney (unpaired) or Wilcoxon (paired) tests for two-group comparisons and a combination of Kruskal–Wallis (interactions and cell type main effect) and Friedman tests (pulse width main effect) with Bonferroni corrected Wilcoxon two-tailed post hoc tests for multi-factor comparisons. Within group variance was compared across the two regions using Levene’s test. In cases of multiple comparisons, p-values for one-sample test, two-group tests or for interaction effects and main effects in multi-factor tests were adjusted using the Bonferroni–Holm correction. Statistical tests were performed using SPSS , VassarStats2 (RRID:SCR_010263) or custom statistics programs . Means are listed with ±standard error of the mean (SEM).All data were tested for normality using the Shapiro–Wilk test. Normally distributed data were compared with the following parametric tests: two-tailed one-sample Student’s To determine whether subiculum Chrna2 cells are a distinct population, Chrna2 cells were characterized according to their Som expression, electrophysiological properties and morphology, and compared to their counterparts in CA1. Post-synaptic responses elicited by Chrna2 cells in subiculum and CA1 pyramidal cells were also characterized and compared. Finally, we aimed to determine whether subiculum pyramidal cells also received EC input, which may be modulated by Chrna2 cells.n = 4; Figure 1Ai). Tom+ and Som+ cells were counted in subiculum and CA1 . In subiculum, 88 ± 1% of Tom+ cells were Som+. In CA1, this proportion was significantly higher at 93 ± 1% . Overall, however, as in CA1, the majority of subiculum Chrna2 cells express Som . The proportion of Som+ cells that are Tom+ was also assessed, and found to be significantly greater in subiculum than in CA1 at 59 ± 2 and 47 ± 3%, respectively . This suggests that Chrna2 cells represent a greater proportion of the Som+ interneuron population in subiculum than in CA1 .To address this first objective, immunohistochemistry experiments were performed with Chrna2-Tom mice , and their resting membrane potentials, responses to depolarizing and hyperpolarizing current steps, and spike properties, were assessed . Chrna2 cells in both subiculum and CA1 fired spontaneously at rest at slow frequency, responded to depolarizing current steps with regular firing, and displayed a sag in their response to hyperpolarizing current steps (Table 1). No significant differences were found for any of the electrophysiological properties examined . Furthermore, there was no significant difference in variance between subiculum and CA1 for any of the electrophysiological properties examined .To assess the electrophysiology properties of subiculum Chrna2 cells, patch-clamp experiments were performed in Chrna2-Tom animals. Chrna2 cells were recorded in subiculum and CA1 . In CA1 Chrna2 cells, the soma and horizontally oriented dendrites were located in the stratum oriens . The axon extended directly out toward SLM, with few collaterals to stratum oriens. Somas for cells in both regions were found to be uniformly ovoid in structure. Like in CA1, the somas of subiculum Chrna2 cells were located in the deepest layer, the polymorphic layer. However, they appeared to be more widely dispersed than in CA1, a feature apparent when visualizing Tom fluorescence in Chrna2-Tom animals . Rather than the narrow band of stratum oriens occupied by Chrna2 cell somas in CA1, the somas of Chrna2 cells in subiculum occupied the full extent of the polymorphic layer, sometimes extending into the deep pyramidal layer. The dendrites of subiculum Chrna2 cells also extended more broadly and gave rise to more extensive dendritic branching than those seen in CA1 . Convex hull analysis showed that the dendritic tree of subiculum Chrna2 cells occupied a significantly greater area than in CA1 . There was no significant difference dendritic tree perimeter  . Sholl analysis showed no significant difference in the number of intersections between Chrna2 cells in subiculum and CA1, but did show a significant effect of radius with proximal and intermediate Sholl segments (20–160 μm) having more intersections than more distal segments  . Axon branching was also more extensive and more variable in subiculum Chrna2 cells than in CA1, with axon collaterals projecting within the polymorphic layer in all cells . For almost all cells, the axons emerged directly from the soma. Unlike in CA1, only four of eight subiculum Chrna2 cells had axons extending superficially to the molecular layer. Additionally, two of eight cells in the subiculum formed local axonal arbors around the soma. This was absent in the CA1. Overall, the soma and dendrites of subiculum Chrna2 cells occupied a broader area than those in CA1, and the axons of subiculum Chrna2 cells extended to both deep and superficial layers, whereas CA1 Chrna2 cells extended primarily superficially.To assess the morphology of Chrna2 cell in subiculum and compare this morphology to CA1, cells were filled with biocytin in patch clamp experiments and reconstructed . Tom+ Chrna2 cells and YFP+ ChETA-expressing cells were counted in subiculum and CA1 . The YFP+ population was assessed to determine the proportion of cells that were either Tom+ or Tom-. The majority of YFP+ cells in both subiculum and CA1  were also Tom+ . These proportions were not significantly different . This suggests that virus expression was specific to Chrna2 cells in both the subiculum and CA1.An optogenetic approach was used to investigate the properties of the post-synaptic responses elicited by Chrna2 cells in CA1 and subiculum pyramidal cells. To activate Chrna2 cells, the blue light sensitive excitatory opsin ChETA was expressed using a cre-dependent viral vector. To first validate opsin expression, the brains of Chrna2-Tom animals injected with AAVdj-EF1α-DIO-ChETA-eYFP were examined using immunohistochemistry  using the same stimulation parameters. Mean photocurrent was found to be -293.5 ± 42.3 pA. The mean minimum pulse width required to elicit a spike was 1.4 ± 0.3 ms and the mean firing frequency over a 500 ms light pulse was 50.4 ± 6.7 Hz. Mean delay between light pulse onset and spike onset was found to be 3.5 ± 0.3 ms. Spike fidelity was robust (mean >90%) for frequencies up to and including 20 Hz (n = 7–9) .It was next determined how efficiently spiking could be activated by blue light pulses in ChETA-expressing Chrna2 cells. YFP+ cells were recorded and responses quantified (n = 39: 29 subiculum (17 regular and 12 burst firing), 10 CA1]. The first aim of these experiments was to compare the responses elicited in CA1, subiculum regular firing or burst firing pyramidal cells. Optogenetic activation elicited inhibitory post-synaptic responses in all three cell types . The amplitude of the inhibitory post-synaptic current (IPSC) was not significantly different between cell types, but did increase significantly with increasing pulse width, with the exception of between 10 and 20 ms . The results were similar when amplitude was measured in cc, with no difference between cell type and with amplitude increasing for every increase in pulse width . To determine whether the kinetics of the response differed between cell types or pulse widths, half width was assessed. The half width of the inhibitory post-synaptic potential (IPSP) was not significantly different between cell types, but did increase significantly with increasing pulse width . Comparison of IPSP decay time yielded similar results with no significant difference between cell type, and a longer decay time for 50 ms pulse than for 5, 10, or 20 ms pulses . To determine if response timing differed between cell types or pulse widths, delay between light onset and IPSP start or IPSP peak was assessed . For both parameters, the delay was not significantly different between cell types . For delay to IPSP start, there was also no significant difference between pulse widths . For delay to IPSP peak, the delay increased significantly with increasing pulse width, with the exception of between 5 and 10 ms . Overall, there was no significant difference in the amplitude, kinetics, or timing of the inhibitory post-synaptic responses elicited by the activation of Chrna2 cells in CA1, subiculum regular firing or subiculum burst firing pyramidal cells.To characterize the post-synaptic responses elicited in pyramidal cells by Chrna2 cell activation, putative pyramidal cells were patched in subiculum and CA1 . IPSCs were also assessed over a series of h.p.’s from -45 to -80 mV to determine the reversal potential . IPSC amplitude decreased from -45 to -80 mV for all h.p.’s, but did not reached 0 or reverse.To further characterize the inhibitory post-synaptic responses elicited by Chrna2 cell activation, paired-pulse recordings were performed over a range of delays to investigate any short-term plasticity in the response . The proportion of cells in which a component remained after gabazine application was greatest for the longest pulse width examined (50 ms) . The smaller amplitude (mean = -0.67 ± 0.07), slower response was completely abolished by subsequent application of CGP-55845, and thus is GABABR mediated. Response amplitude, kinetics and timing were assessed to examine the GABABR-mediated component in further detail and to determine whether it might be different between subiculum regular and burst firing and CA1 pyramidal cells . IPSP amplitude was significantly smaller after gabazine application compared to control conditions, but was not significantly different between cell types . Response half width  and decay time were significantly longer after gabazine application compared to control conditions, but were not significantly different between cell types . Delay from light onset to IPSP start  and to IPSP peak were also significantly longer after gabazine application compared to control conditions, but was not significantly different between cell types . Overall, the GABABR-mediated component of the post-synaptic response elicited by Chrna2 cell activation is similar in amplitude, kinetics and timing for CA1 and subiculum regular and burst firing pyramidal cells.To determine what receptors may mediate the post-synaptic response to Chrna2 optogenetic-cell activation, responses were recorded after bath application of gabazine, a GABAChrna2 was recently discovered to be a specific genetic marker for CA1 OLM interneurons , interneChrna2 cells in the subiculum were characterized according to their Som expression and electrophysiological and morphological properties, and these properties were compared to those of Chrna2 cells in CA1. The majority of subiculum Chrna2 cells expressed Som, as did those in CA1. This finding is consistent with the characteristic Som expression associated with OLM interneurons in CA1 and CA3 . In subiVr, regular spiking with some frequency accommodation in response to depolarizing current steps, and a sag in their responses to hyperpolarizing current steps (Ih current, a hyperpolarization-activated conductance believed to play a role in the theta frequency firing recorded in OLM interneurons (The electrophysiological properties of Chrna2 cells in the subiculum and CA1 were homogeneous and not significantly different. Chrna2 cells in both sub-regions displayed characteristic features of CA1 OLM interneurons: slow, spontaneous firing at nt steps . The sagrneurons .Finally, the morphology of Chrna2 cells in subiculum and CA1 was characterized and compared. CA1 Chrna2 cells displayed a morphology that was similar to that described in previous studies of CA1 OLM interneurons . SubiculAn optogenetic technique was used to activate Chrna2 cells and post-synaptic responses were recorded in CA1 pyramidal cells as well as from subiculum regular and burst firing pyramidal cells. Responses in both regions were inhibitory, as expected from a previous study in CA1  and the Paired-pulse recordings revealed modest paired-pulse depression, consistent with previous work in CA1 . This suARs (AR conductances generated in distal dendrites due to poor vc at these distal sites The response may also involve GABABRs, which have a potassium-mediated reversal potential at closer to -90 mV (AR antagonist gabazine attenuated a significant component of the response, but a smaller amplitude, slower component, consistent with a GABABR-mediated response, remained (BR antagonist CGP-55845. This study is the first to show a GABABR component in OLM interneuron post-synaptic inhibition. This GABABR-mediated response was significantly smaller in amplitude and displayed slower kinetics and longer delays to response start and peak. No significant differences were found in amplitude, kinetics, or timing between cell types, confirming that Chrna2 cells elicit similar responses in both CA1 and subiculum and both subiculum pyramidal cell types. Overall, these results suggest that Chrna2 cell-mediated post-synaptic responses in both subiculum and CA1 are composed of both fast GABAAR and slow GABABR mediated components.Post-synaptic responses were also recorded at h.p.’s from -45 to -80 mV to determine their reversal potential. The inhibitory response decreased as the h.p. was more negative, but did not reverse. This was surprising as the range of h.p.’s examined were close to the reversal potential for chloride, the ion conductance associated with GABAARs . This mao -90 mV . In the remained . This reApart from some morphological differences, the results of this study indicate that subiculum Chrna2 cells are generally similar to those in CA1, particularly in their effects on pyramidal cells. These similarities suggest that they may also serve similar roles. Two areas of particular interest are roles in theta rhythm and memory function.OLM interneurons in CA1 are believed to play a role in the modulation of theta oscillations. They have been found to increase their firing during theta rhythm and phase lock to the trough of the theta cycle . RhythmiOLM interneurons in CA1 are also believed to play a role in memory. ARs and GABABRs, and are likely to play similar roles both sub-regions, roles which may include the regulation of theta rhythm and a contribution to learning and memory functions.Overall, this study has provided the first in-depth characterization of Chrna2 cells in the subiculum and has enhanced our understanding of how this interneuron population may contribute to subiculum function. The findings suggest that subiculum and CA1 Chrna2 cells are largely equivalent, modulate pyramidal cell activity through GABAHN was the primary contributor to project design, data acquisition, analysis and interpretation, and manuscript preparation. SW was the primary contributor to project conception and significantly contributed to project design, data interpretation, and critical revision. BA, FM, and SB contributed significantly to data acquisition, analysis and interpretation, and critical revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.