Connectivity determines the function of neural circuits and it is the gateway to behavioral output. The emergent properties of the Auditory Cortex (ACx) have been difficult to unravel partly due to our assumption that it is organized similarly to other sensory areas. But detailed investigations of its functional connectivity have begun to reveal significant differences from other cortical areas that perform different functions. Using Laser Scanning Photostimulation we previously discovered unique circuit features in the ACx. Specifically, we found that the functional asymmetry of the ACx (tonotopy and isofrequency axes) is reflected in the local circuitry of excitatory inputs to Layer 3 pyramidal neurons. In the present study we extend the functional wiring diagram of the ACx with an investigation of the connectivity patterns of inhibitory subclasses. We compared excitatory input to parvalbumin (PV) and somatostatin (SOM)-expressing interneurons and found distinct circuit-motifs between and within these subpopulations. Moreover, these connectivity motifs emerged as intrinsic differences between the left and right ACx. Our results support a functional circuit based approach to understand the role of inhibitory neurons in auditory processing. In a previous study we compared the connectivity between orthogonal functional axes in the Auditory Cortex (tonotopy and isofrequency) and found unique circuit-motifs in excitatory networks along the tonotopic axis that were absent along the isofrequency axis. These findings suggest that auditory cortical microcircuitry is specialized to the unique one-dimensional representation of frequency in the Auditory Cortex (ACx)1. Other studies have also challenged the idea that cortical circuits are homogeneous processing modules with identical flow of information4. Compared to the visual and sensorimotor cortices, the connectivity in the Auditory Cortex is not as well understood. The full connectivity of most auditory circuits and their role in sound perception remain unknown. Here, we continue to fill this gap by mapping the functional synaptic connectivity of two genetically identified inhibitory cell classes.The cerebral cortex guides behavior by extracting salient sensory signals. These functions are executed by cortical modules that share many features  but also contain significant specializations. By relating detailed connectivity information to sensory coding 5. Nevertheless, three genetically defined sub-classes have dominated recent studies of inhibitory function thanks to readily available cre-driver transgenic mouse lines6. Here we compare the connectivity of two sub-classes: parvalbumin (PV) and somatostatin-positive (SOM) inhibitory neurons. In addition to being molecularly and morphologically distinct, these subclasses also employ different mechanisms to control neural activity. Using cre-driver lines and optogenetic tools, researchers have begun to dissect the function of these cell classes in neural computations. Some of their proposed functions include shaping receptive fields7, gain control, and surround suppression9. However, in the ACx the laminar and cell-type dependent tuning properties of excitation and inhibition remain under debate10. The lack of knowledge of the functional connectivity of inhibitory neurons in the ACx has hindered our ability to build detailed models or reject existing models of their role in auditory function. The diverse circuit-motifs of inhibitory neurons in other cortical areas supports the need for this information in the ACx12. Although molecular classification schemes are helpful, circuit function is a more useful way to classify neurons13. In support of this proposal, we found distinct circuit-motifs in the connectivity of PV and a sub-population of SOM inhibitory neurons in the left and right ACx. These findings underscore the need for connectivity-based and region-specific insight of inhibitory microcircuits in cortex.Inhibitory interneurons are the most diverse cell-type in cortex whose task is to control the flow of activity within local circuits. Interneurons can be classified according to molecular, morphological, and biophysical properties, though with many ambiguities3, 25 D-glucose, 11.6 sodium ascorbate, 7 MgCl2, 3.1 sodium pyruvate, 2.5 KCl, 1.25 NaH2PO4, and 0.5 CaCl2. We made 300 μm thick horizontal slices cut at a 15-degree angle between the blade and the medial-lateral axis of the brain14. This preparation preserves tonotopy: the anterior-posterior representation of frequencies in ACx. Slices were then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 127 NaCl, 25 NaHCO3, 25 D-glucose, 2.5 KCl, 1 MgCl2, 2 CaCl2, and 1.25 NaH2PO4, aerated with 95% O2 5% CO2. The slices were incubated at 34° for 20–30 minutes and then kept at room temperature during the experiments. The ACx lacks clear anatomical landmarks to align the stimulus grid for each cell, which is essential to establish consistent tonotopic position across animals. To overcome this challenge, we developed a systematic coordinate system. We center the x-axis of the photostimulation grid on the soma of the neuron recorded and align the y-axis with the second row of the grid on the L1/2 border, the clearest laminar boundary in our slices. For each cell, we measure spatial coordinates: distance from the soma to the pial surface, L1/2 border, and the horizontal distance to the anterior tip of the hippocampus (where the fimbria exits). The latter measurement is used to assign each cell a position along the anterior-posterior tonotopic axis  approved by the Institutional Animal Care and Use Committee (IACUC) of Cold Spring Harbor Laboratory. To visualize inhibitory cell types, we bred PV and SOM cre-lines with the Ai14-tdTomato reporter mouse (Jackson labs), and combined differential interference contrast microscopy with fluorescence to guide whole-cell recordings Fig. . Male mixis Fig. .Figure 12, 10 HEPES, 1 EGTA, 4 Na2ATP, 0.4 Na2GTP, and 10 Na-phosphocreatine; pH 7.25, 300 mOsm). The recording artificial cerebrospinal fluid contained (in mM) 0.37 nitroindolinyl (NI)-caged glutamate (Tocris), 0.005 CPP (Tocris), 4 CaCl2 and 4 MgCl2. The high divalent concentration and CPP (NMDA channel blocker) prevent reverberant excitatory activity due to photostimulation. Voltage-clamp recordings were made using a Multiclamp 700 A amplifier . We photoreleased the caged glutamate compound with a 1-ms light stimulus consisting of 100 pulses from a pulsed UV laser . To map hotspots of presynaptic input we used a 16 × 16 stimulus grid with 75 μm spacing, resulting in a mapping region of 1.125 × 1.125 mm. Hotspots in the maps correspond to areas that connect monosynaptically to the recorded cells.We performed fluorescence-guided patching of inhibitory neurons 45–80 μm below the surface of the slice visualized using infrared gradient contrast optics with electrodes (6–7 MΩ) containing the following intracellular solution (in mM) 128 K-methylsulfate, 4 MgCl1. Briefly, the mean current amplitude of Excitatory Postsynaptic Currents (EPSCs) was calculated in the 50 ms epoch after the direct response time window /(Anterior Input + Posterior Input)An asymmetry index of 0 would indicate synaptic input centered on the soma of the cell mapped, whereas values between 0 and ±1 would indicate spatially/tonotopically shifted synaptic input.All raw data and basic analysis software will be made available upon request to the author.16. To isolate excitatory input onto these interneuron subclasses, we voltage-clamped cells near the inhibitory reversal potential (−70 mV).We previously characterized the connectivity of Layer 2 and 3 pyramidal neurons and found distinct connectivity patterns between these populations of principal neurons in horizontal slices of the ACx that preserve tonotopy. Because we found out-of-column circuit-motifs unique to the ACx in excitatory populations, we now focused on mapping the connectivity of inhibitory neurons in L3 of the ACx. Furthermore, we explored the potential for hemispheric differences in connectivity given the hypothesized functional division of labor between the left and right ACx. To target genetically defined inhibitory subclasses, we used two well-established transgenic mouse lines that express cre-recombinase in parvalbumin-expressing (PV), and somatostatin-expressing (SOM) inhibitory neurons. Together with vasoactive intestinal peptide-expressing (VIP) cells, these markers account for 84% of all inhibitory neurons in cortex and are believed to capture largely non-overlapping, molecularly-defined populations17. A representative map of the average presynaptic input for a PV cell in L3 of the left ACx is shown in Fig. 12, and using pair-recordings in the ACx18.PV-expressing interneurons make up the largest population of inhibitory neurons. They tend to be fast-spiking, and target the cell body and axon hillock of pyramidal neurons. Although this subclass includes chandelier cells, PV-cre transgenic lines typically only capture basket cells. Furthermore, basket cells have been further sub-divided based on distinct patterns of axonal arborizationp = 0.03, ranksum n = 21 from 6 mice; Fig. p = 0.0042, ranksum n = 21 from 6 mice; Fig. p = 0.78, L2: p = 0.85, L3: p = 0.45, L4: p = 0.32; ranksum n = 21 from 6 mice; Fig. L5 to L3 = 0.1354 and L6 to L3 = 0.2122, n = 11 cells from 3 mice).To determine if a similar pattern of recurrent and L4 feedforward connectivity is conserved in the right ACx, we mapped a population of Layer 3 PV cells in horizontal slices. Unexpectedly, we found a significantly different circuit-motif in the right ACx. A representative input map shows that in addition to recurrent and feedforward input from L4, there is substantial infragranular input arising from L5/6 Fig. , top. Th19. We mapped the connectivity of SOM interneurons in L3 of the left and right ACx and found distinct inter- and intrahemispheric circuit-motifs. In the right ACx we found two patterns of connectivity embedded in the same layer. Approximately half of the population of SOM interneurons received input from L6 . Synaptic input from other layers was not significantly different between these two populations . Similar to PV cells in the right ACx, the spatial/tonotopic organization of the L6 to L3 input was not centered on the somata .Comparable to PV-positive cells, somatostatin (SOM)-expressing inhibitory interneurons are not a monolithic population; they can be further subdivided by molecular markers, firing patterns, morphology and synaptic targetsata Fig. , top. Mop = 0.71, L2: p = 0.97, L3: p = 0.87, L4: p = 0.17, p = 0.06, p = 0.08, ranksum n = 23 cells from 8 mice).In contrast to the two distinct circuit-motifs observed in the right ACx, in the left ACx all the SOM-expressing cells mapped only received feedforward input from L4 and recurrent input from L2/3 . The asymmetry indices of all the infragranular input pathways are summarized in Fig. Finally, we compared synaptic input across cell classes and hemispheres Fig. . We foun1. In the present study we uncovered distinct circuit-motifs between subpopulations of inhibitory neurons in L3 within and between the left and right ACx (Fig. Previously we discovered that the asymmetric nature of frequency representation in the ACx is reflected in the connectivity of excitatory neurons in the ACx (circuit diagram in Fig. ACx Fig. . Mainly,17. At the same time, genetic tools have come online to target broad classes of interneurons to facilitate the study of their function11. In addition to their diversity, the distribution of specific subclasses varies between different areas. For example, there are more PV interneurons in primary sensory regions than elsewhere in the cortex20. An unresolved question has been whether specific circuit-motifs vary between cortical areas as well.The molecular, morphological, and electrophysiological diversity of inhibitory neurons have been their hallmark for decades. In fact, recent efforts have focused on finding parameters to reduce the diversity of their classification. One approach is to use clustering algorithms to develop an unbiased and standard taxonomy23. These results are distinctly different to the functional connectivity we report here in the right ACx using the same circuit mapping technique. Nevertheless, there is evidence of pathway-specific inhibitory microcircuits within the somatosensory cortex, where several subclasses receive differential input from lemniscal and paralemniscal pathways12.Previous connectivity studies using LSPS have revealed the largely local nature of inhibitory neural connectivity. LSPS studies in the visual and barrel cortices showed that several classes of inhibitory neurons in L2/3 receive strong feedforward input from L4 and recurrent connections within L2/3; whereas input from L5 is weak, and none arises from L624. This could be in part because the three cre-driver lines used with optogenetic tools target functionally heterogeneous subpopulations of interneurons19. Our results underscore some of the shortcomings of using these tools: within the same layer we discovered distinct connectivity patterns for the same genetically defined subclass. Although genetic tools are useful and will continue to improve, our results support that circuit-specific function motifs will be a more useful paradigm to classify inhibitory neurons13.The specific function of inhibitory interneurons in neural computations is still under debate25. In contrast, the L5/6 excitatory projections we discovered in the right ACx directly target interneurons in superficial layers. On the other hand, connectivity in the left ACx for both inhibitory cell types we examined is more in line with findings in the visual and barrel cortices: feedforward input from L4 and recurrent connections within L2/3 (Fig. 10, whereas others report systematic differences across layers and cell-types27. Moreover, PV interneurons in L2/3 appear to be more broadly tuned than somatostatin7. Our results suggest that in addition to subclass coding differences, we also need to strongly consider intralaminar and hemispheric circuit-motifs in our studies and interpretation of inhibitory function in the ACx.In the visual cortex, the gain of visually-driven responses has been found to be modulated via the activation of L6 pyramids that target neighboring PV interneurons with axons that arborize throughout all layers, leading to suppression of an entire cortical column2/3 Fig. . In the 28. The efficiency of decoding rapidly changing sounds is improved by hemispheric lateralization, as found for human speech30. This adaptation is also widespread in other animals, including mice, suggesting their auditory cortex may share mechanisms that can be studied using the powerful circuit-analysis tools available in this species. Functionally, the left and right ACx are postulated to specialize in processing different spectro-temporal features of sounds, but the mechanisms remain unresolved30. The left ACx is believed to preferentially process vocalizations32, whereas behavioral studies in rodents have shown a processing bias to detect the direction of frequency-modulated sweeps in the right ACx by using global cues33. Further, the right ACx of the rat has a sweep direction selectivity map in parallel with the tonotopic map34. In future studies we will determine whether the inhibitory connectivity motifs we found in this study play a role in sweep direction selectivity. One clue to their function may lie in the predominant tonotopic bias of the L5/6 input: most hotspots were not centered on the soma of the cell recorded (Figs One proposed function of the Auditory Cortex is to form neural representations of ethologically relevant, spectrotemporally complex stimuli like animal vocalizationsded Figs , 4A, 5A.


The transition from the last ice age to the present-day interglacial was interrupted by the Younger Dryas (YD) cold period. While many studies exist on this climate event, only few include high-resolution marine records that span the YD. In order to better understand the interactions between ocean, atmosphere and ice sheet stability during the YD, more high-resolution proxy records from the Arctic, located proximal to ice sheet outlet glaciers, are required. Here we present the first diatom-based high-resolution quantitative reconstruction of sea surface conditions from central-eastern Baffin Bay, covering the period 14.0–10.2 kyr BP. Our record reveals warmer sea surface conditions and strong interactions between the ocean and the West Greenland ice margin during the YD. These warmer conditions were caused by increased Atlantic-sourced water inflow combined with amplified seasonality. Our results emphasize the importance of the ocean for ice sheet stability under the current changing climate. High-resolution proxy records are essential for understanding the interactions between ocean, ice sheet and atmosphere. Here the authors present a high-resolution record of Younger Dryas sea surface conditions in Baffin Bay and show that ocean surface warming triggered the retreat of Jakobshavn Isbræ ice stream. Although the trigger of the event remains a matter of debate, it has been frequently associated with fresh water release into the North Atlantic4. The resulting stratification would have weakened the Atlantic Meridional Overturning Circulation (AMOC), which is closely coupled to climate fluctuations5, and disrupted heat transport to high latitudes6. However, a recent modeling study found that fresh water release alone could not have caused the cold period, but additional triggers were required7. The YD had a stronger impact on the Northern Hemisphere, although signs of this colder period have also been detected in tropical paleo-records8.The Younger Dryas (YD) cold period at 12.9–11.7 kyr BP9 and accelerated ice mass loss from the Greenland Ice Sheet (GrIS)10, there is a need for high-resolution marine proxy data to better understand the interactions between the ocean and the outlet glaciers of the GrIS. So far, high-resolution studies on YD ocean surface conditions based on marine records from the Arctic region are rare12. Baffin Bay 17 underlain by a layer of colder water down to ca. 100 m wd, below which temperature and salinity increase, yet remain slightly colder than the surface waters  and the warmer West Greenland Current (WGC). The WGC consists of polar waters from the East Greenland Current (EGC) and of Atlantic-sourced waters from the Irminger Current (IC) Fig. . The EGC20 and in the West Greenland sector ice reached the outer shelf18. Although the timing and the pattern of the deglaciation around Baffin Bay is poorly constrained, it has been suggested that the retreat of West Greenland ice streams was asynchronous21 and occurred between 17 and 11.5 kyr BP22. One of the most significant known retreats on the West Greenland margin was the collapse of the Jakobshavn Isbræ ice stream into Disko Bay during the YD23. Today the Jakobshavn Isbræ is one of the largest ice streams in Greenland, draining about 7% of the GrIS area24 and producing 10% of the total iceberg discharge from the GrIS25. It has been proposed that ice stream retreat in the east, south−east, south−west, and west of Greenland may have been triggered by the incursion of warmer waters during the YD29, and that the ice margin retreat was out of phase with atmospheric temperatures over Greenland. These fast-flowing ice streams are strongly influenced by sea surface temperatures (SSTs) at their front31 and contribute today one-third32 of the yearly ice loss from the GrIS.During the LGM, ice margins from the northeastern Laurentide Ice Sheet (LIS), southern Innuitian Ice Sheet (IIS) and western Greenland Ice Sheet (GrIS) entered the Baffin Bay33, which is based on a large northern North Atlantic calibration data set34. Q-mode factor analysis, using the same calibration data set, was also conducted to relate modern diatom assemblages to different ocean water masses and applied downcore to assess changes in the relative influence of different water masses off West Greenland during the studied time period. The proportion of the bulk sediment in the clay and silt (<63 µm) size fraction was used as a proxy for the influence of the glacier meltwater suspension plume35, as size fractions larger than this are too large to be carried in the plume36. This high-resolution reconstruction of sea surface conditions in Baffin Bay during the deglacial shows warmer SSTs during the YD, and indicates these played a key part in the Jakobshavn Isbræ ice margin retreat. We conclude that these warmer conditions were caused by an enhancement of Atlantic-sourced water inflow together with increased seasonality. Based on our data, the retreat of the Jakobshavn Isbræ during the cold YD occurred mainly by calving, while surface/subsurface melt dominated during a warmer climate before and after the YD.Here we use the high-resolution sediment core SL 170, strategically located close to the maximum post-LGM extent of the Jakobshavn Isbræ  used in this study represents the age range from ca. 14.0 to 10.2 kyr BP —after a short-lived YD advance , which would have thinned the outlet and made it vulnerable to collapse—would agree with the scenario proposed by Jennings et al.39. The exact behavior of the Jakobshavn Isbræ retreat after the collapse is unclear and two alternatives, either a sustained retreat39 or a second collapse at ca. 10.2 kyr BP23, have been proposed.Modern Jakobshavn Isbræ calving is known to be strongly affected by warmer Atlantic waters from the Irminger Sea14C reservoir age (ΔR) for radiocarbon chronologies is challenging due to its variation through time, thus we addressed this issue by testing a range of ΔR corrections 20 indicating meltwater and iceberg discharge in Baffin Bay and the Labrador Sea region. Due to its location and exceptionally high resolution the record facilitates direct comparison with the known behavior of Jakobshavn Isbræ39, and provides strong support for warmer sea surface conditions that is suggested as a principal driving mechanism in the hypothesis of Rinterknecht et al.23. The reconstructed aSSTs show an obvious mismatch with the δ18O record from the North Greenland Ice Core Project (NGRIP)1 1 Fig.  and with39, the bulk of IRD is typically lost relatively close to the source in particular under warmer ocean conditions44. Our data indicate that warming ocean surface conditions led to intensive calving and retreat of the Jakobshavn Isbræ beginning already around 13.4 kyr BP. Sedimentation rates are markedly higher between ca. 13.2 and 11.7 kyr BP, while the decreasing amount of fine sediments from the meltwater suspension plume imply increasing distance from the retreating glacier35. The high variability in the Arctic Water and MIZ assemblages between 12.5 and 12.1 kyr BP indicates strongly fluctuating surface water conditions between fresh meltwater and Arctic water suggesting that calving and associated melting of icebergs occurred in distinct episodes.Instead of recording a cooling of the ocean surface during the YD cold period, the reconstructed aSSTs show warmer conditions (compared to aSSTs before and after YD) starting at ca. 13.4 kyr BP and continuing to the end of the YD Fig. . The hig21, who concluded that the retreat of Jakobshavn Isbræ was modulated only by topography and ice dynamics rather than climatic or oceanic drivers, we propose that both the YD re-advance and subsequent retreat were governed by a combination of an oceanic driver and topographic control21. Here, the advance would represent a surge leading to ice thinning, which in turn caused the rapid retreat (collapse) documented in previous studies39. The uplift adjustment used in Rinterknecht et al.23 did not take into account the gravitational attraction of water toward ice . If this is accounted for, the collapse would have occurred at the end of the YD, when we see a marked drop in sedimentation rates indicating that the glacier had retreated away from the outer shelf . Instead, increased seasonality, as indicated by our reconstructed higher summer temperatures, with open water conditions , and at 5 cm intervals from the depths of 195–475 cm (ca. 14.0–11.9 kyr BP). Grain size distribution was measured from 77 samples at ca. 50 years resolution using a Malvern Mastersizer 2000(G) particle size analyser.The marine sediment core SL 170 was recovered from the deepest ploughmark on the Disko Trough outer shelf in central-eastern Baffin Bay  picked from the >150 µm fraction, and two mollusc fragments were also dated  indicate that miliolid spp. were scarce downcore. On the rare occasion when miliolid spp. (which are known for giving too old ages) were found, they were not included in the samples sent for radiocarbon dating.Twenty-one Accelerated Mass Spectrometry (AMS) 55 using the Marine13 calibration curve56 and a local reservoir correction (ΔR) of 140 ± 35 years57 as has been used in previous studies in Disko Bay e.g., refs 39. Bacon software58 was applied to create an age-depth model , and clay separation59. A minimum of 300 diatom valves were identified at each level using ×1000 magnification60. Chaetoceros resting spores are routinely excluded from the total diatom counts because they generally completely dominate fossil assemblages yet they show negligible sensitivity to SST61. aSST was reconstructed using a weighted-averaging partial least squares (WA-PLS) transfer function33 based on a modern calibration data set34 consisting of 183 surface sediment samples from the North Atlantic, including the Labrador Sea, the Nordic Seas, and Baffin Bay. The 2-component WA-PLS model has a root mean square error (RMSE) of 1.14 °C, r2  of 0.92 and a maximum bias of 2.81 °C. The prediction error was estimated using h-block cross validation62 in which samples closer than a cutoff distance (h) from a target sample were excluded from contributing to the prediction of that sample. h-block cross validation was selected to allow for spatial dependency in the calibration data, which can lead to underestimation of the prediction error because of pseudoreplication. The cutoff distance (h) was estimated by assessing the spatial structure of the residuals of the surface sample predictions. Specifically, h was estimated as 495 km using the range of a circular variogram fitted to the detrended residuals63. The optimal number of WA-PLS components was determined using a randomization t-test62 applied to h-block prediction errors.Diatom samples were prepared using hydrogen chloride (HCl), hydrogen peroxide  that are related to distinct ocean water masses; Marginal Ice Zone, Arctic Waters, Greenland Arctic Waters, East−West Greenland Current, Transitional Waters, Sub-Arctic Waters, North Atlantic Current, and Norwegian Atlantic Current . The factor model can then be applied downcore to reconstruct changes in the relative importance of the various assemblages (and hence water masses) through time. More specifically, given the M by N row-normalized matrix Ucd constructed of m = {1,…, M} calibration data (“cd”) set samples of n = {1,…, N} diatom species each, the results of the Q-mode factor analysis are formulated as:Bcd is an M by L varimax rotated factor loadings matrix, F is a factor scores matrix of size L by N, E is an error matrix, and L < N denotes the number of factors retained for further analysis. Each element lm of Bcd, l = {1,…, L}, represents a proportional contribution of factor l into calibration data set sample n. In turn, F describes the species compositions of L varimax rotated factors: each element Fln of F reflects the relative “importance” of species n in factor l.A Q-mode factor analysisUc of downcore diatom assemblages on transposed F:K by L matrix Bc of the decomposition of K downcore samples into previously defined L varimax factors. Samples communality, found in the main diagonal of matrix BBt indicates an adequacy of the derived decomposition in terms of the accounted variance.Projecting the row-normalized matrix 65 provide a detailed description of the eight factors (assemblages) and their spatial distribution. The name of the Sea Ice assemblage in Andersen et al.65 was re-named to MIZ assemblage in this study as the dominant indicator species in the assemblage, Fragilariopsis oceanica (Cleve) Hasle and Fragilariopsis cylindrus (Grunow) Krieger in Helmcke and Krieger, dwell in the cold, fresh surface water layer that is produced by melting glacier ice/sea ice. This name reflects more adequately the true (still sea ice related) ecology of these species. These species inhabit both sea ice and open water, but typically form blooms in the melt layer overlying ambient sea water68. Hence the MIZ assemblage can be used as an indicator of past melt periods.Andersen et al.http://www.ncdc.noaa.gov/paleo; https://www.pangaea.de).All data presented in this paper are available in the NOAA paleoclimate database and in the open database for Earth and Environmental Science PANGAEA (Supplementary InformationPeer Review File


More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross-reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope–derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergen-specific Ab responses were subsequently boosted with molecules containing the B cell epitope–derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines. Grass pollen allergens are the most frequent elicitors of IgE-mediated allergies worldwide, provoking allergic symptoms such as rhinitis, conjunctivitis, allergic asthma, and atopic dermatitis –5. In EuIn this study, we investigated in a mouse model to which extent B cell and T cell epitopes derived from the major grass pollen allergen Phl p 1 are involved in boosting secondary IgE responses. We prepared a recombinant fusion protein consisting of a major IgE epitope–containing portion of Phl p 1 and of an allergen-unrelated viral carrier protein, the PreS surface protein from hepatitis B, for induction of IgE sensitization toward Phl p 1 . In thisA 30-aa peptide derived from the major grass pollen allergen Phl p 1 ranging from aa 212 to 241 (VRYTTEGGTKTEAEDVIPEGWKADTSYESK) was synthesized using a Wang resin from AAPPTec  on a Liberty microwave peptide synthesizer from CEM . Additionally, the same peptide was prepared containing an N-terminal cysteine residue as previously described . For conPurified synthetic peptides were analyzed by means of SDS-PAGE under reducing and nonreducing conditions. Nonreducing conditions were obtained by dissolving the peptides in sample buffer without 2-ME. Samples were separated in 18% SDS-PAGE without boiling. Gels were subsequently stained with Coomassie blue .c] = 1 μg/μl; dimeric peptide [2xP], c = 1 μg/μl) were loaded onto a Superdex peptide 10/300 GL column  at room temperature and equilibrated with 10 mM phosphate buffer (pH 7.4) containing 137 mM NaCl. The flow rate was 0.5 ml/min, fractions of 1 ml were collected, and the absorbance was measured at 280 nm. Fractions showing a peak were further analyzed by MALDI-TOF. Dextran blue (2000 kDa), a peptide dimer (7.8 kDa), and a peptide monomer (3.9 kDa) were used as standards.For gel filtration, 300-μl aliquots of the peptides  was chosen for this study owing to the lack of Phl p 1–specific T cell epitopes recognized by BALB/c mice  27). Mo. Mo27). Circular dichroism (CD) measurements of purified peptides 1xP and 2xP dissolved in water were performed on a Jasco J-810 spectropolarimeter  using a 1-mm quartz cuvette. Spectra were recorded at a scan speed of 50 nm/min at room temperature and were corrected by subtracting the corresponding baseline spectrum, which was measured for water at identical conditions. Results are means of three measurements and are expressed as molecular ellipticity .c = 1 μg/μl in water), 1 μg of 2xP (c = 1 μg/μl in water), 2.2 μg of 4xP , 1 μg of oligo-P, calculated according to the coupling efficiency (c = 1 μg/μl in PBS)], PreS and KLH in equimolar amounts as contained in 4xP and oligo-P ), 1 μg of KLH, calculated according to the coupling efficiency (c = 5 μg/μl in water)], and 1 μg of an irrelevant Ag  for control purposes were dotted onto nitrocellulose membrane strips , air-dried, and blocked with 3% nonfat dry milk powder  for 2 h at room temperature. The strips were then incubated at 4°C overnight with respective rabbit antisera  diluted 1:10,000. Bound Abs were then detected with 1:5000 diluted [125I]-labeled goat anti-rabbit IgG Ab  and visualized by autoradiography.The proteins 1xP, 2xP, 4xP, and oligo-P, each representing 1 μg of peptide (P) [i.e., 1 μg of 1xP  and kept in the animal care unit of the Department of Pathophysiology and Allergy Research, Medical University of Vienna  according to the local guidelines of laboratory animal care. All animal experiments were approved by the local Ethics Committee.3  on day 1. Blood samples were collected from tail veins of the mice on day 0 (preimmune serum) and on day 22 after sensitization (immune serum) to investigate the induction of peptide P–specific IgE responses. A group of mice (n = 5) was sensitized and then boosted three times by s.c. injections of Al(OH)3-adsorbed 4xP (44 μg) in 4-wk intervals. On day 180, spleens were removed under aseptic conditions for preparation of splenocytes and analysis of T cell responses by violet proliferation dye (VPD)450 FACS experiments and thymidine incorporation assays. Sera from these mice obtained on day 180 were later used to sensitize rat basophilic leukemia (RBL) cells for testing the molecules used for boosting.In a first pilot experiment, we studied whether IgE Abs and an allergic immune response could be induced against peptide P by sensitization with 4xP. Eight mice were sensitized with an s.c. injection of 44 μg of 4xP  adsorbed to Al(OH)3-adsorbed 2xP  and boosted on day 165 with an s.c. injection of 2xP (20 μg of P) in PBS without adjuvant. Peptide-specific IgE and IgG Ab responses were analyzed in blood samples obtained on days 0, 7, 14, 28, 42, 164, 172, and 179 by ELISA.In a second set of experiments we studied whether sensitization with peptide P  without carrier will induce peptide P–specific Ab responses. For this purpose, an additional five mice were sensitized on day 1 with an s.c. injection of Al(OH)n = 5) were sensitized s.c. on day 1 with 44 μg of 4xP  adsorbed to Al(OH)3. A control group  received only PBS and Al(OH)3 (n = 8) and results were added in In a third set of experiments, we studied whether IgE and IgG responses induced by sensitization with 4xP can be boosted with different immunogens.  Al(OH)3 . Blood sn = 5), labeled with VPD450  according to the manufacturer’s instructions, and cultivated for 4 d in the presence of peptide 1xP (0.3 μg/well), the carrier molecule PreS (1 μg/well), Con A (0.5 μg/well), or medium. Subsequently, cells were stained with FITC-labeled rat anti-mouse CD4 Ab  or isotype control . Cells were analyzed on a FACSCanto . Data of flow cytometry were analyzed in FlowJo version 10 (Miltenyi Biotec). Lymphocytes were gated in forward and side scatter to remove cell debris. Lymphocytes were further gated for CD4+ cells and analyzed for proliferation rates of CD4+ and dilution of VPD450 after stimulation as indicated. Representative blots of stimulated splenocytes of one mouse are demonstrated  diluted 1:6000. Bound IgG Abs were detected with rat anti-mouse IgG1 mAb  diluted 1:1000, followed by an HRP-conjugated goat anti-rat antiserum  diluted 1:2000. The specificity of the goat anti-mouse IgE antiserum for mouse IgE was confirmed by ELISA, as it detected mouse monoclonal IgE  were coated with 2xP (5 μg/ml), blocked with 1% (w/v) BSA in PBST for 2 h, and incubated with mouse sera diluted 1:10 for IgE and 1:500 for IgGonal IgE  but not E  for 20 h at 37°C using 5% CO2.RBL cell mediator release assays were carried out as described . RBL-2H3n = 8) was tested by loading individual sera (1:10 diluted) for 2 h on RBL cells and by induction of mediator release adding 0.3 μg/ml peptide–OVA conjugate.Sensitization of 4xP-immunized mice  to remove unbound Abs. Cells were exposed to each immunogen containing 0.3 μg/ml peptide P. β-Hexosaminidase release was analyzed after 1 h.To test the ability of the different immunogens  to cross-link peptide P–specific IgE Abs on basophils, RBL cells were loaded with 1:10 diluted pooled sera obtained on day 180 from mice sensitized and boosted three times with 4xP. Cells were then washed twice with Tyrode’s buffer , irrelevant peptide (0.3 μg/well), PreS (2 μg/well), or Con A (0.5 μg/well) as positive control. The cultures were then pulsed with 0.5 μCi/well tritiated thymidine for 16 h and harvested. The proliferation responses were measured by scintillation counting on a Wallac MicroBeta TriLux scintillation counter (PerkinElmer). The ratio of the mean proliferation after Ag stimulation and medium control values, that is, the stimulation index (SI), was calculated.Spleens were removed under aseptic conditions (day 185) and homogenized. After lysis of erythrocytes, cells were washed and resuspended in supplemented medium . Single-cell suspensions were plated into 96-well round-bottom plates at a concentration of 2 × 10p value < 0.05 was considered statistically significant. Results are either shown as box plots with indicated medians using SPSS software version 20.0 or column bar graphs using GraphPad Prism (GraphPad Software) version 5.0.Comparison of Ab levels within each mouse group was performed by the Wilcoxon signed rank test for paired data using SPSS software. A To investigate the contribution of B and T cell epitopes on secondary allergen-specific IgE Ab responses, we followed the hapten-carrier principle established by Benacerraf and constructed a fusion protein consisting of a peptide from the IgE epitope–containing region of the major timothy grass pollen allergen, Phl p 1, and an unrelated carrier protein  providing T cell help  24)..24).The Phl p 1 peptide P comprised 30 aa of the C terminus of Phl p 1  where the major IgE epitopes of allergic patients are located  Fig. 1A, gray. CPure monomeric peptide P  was obtained by chemical synthesis, and its experimental molecular mass determined by mass spectrometry   and was Escherichia coli and purified , in the recombinant fusion protein (4xP), and in the KLH conjugate (oligo-P) in their native form by dot blotting . Dot-blo+ T cells could only be detected in response to the carrier , but not in response to the peptide , which was in the range of the medium control . Con A–stimulated cells showed a mean proliferation of 68.56% . These results show that immunization of mice with 4xP induced a peptide-specific IgE Ab response without detectable peptide-specific T cell help whereas carrier-specific T cells were detected.In a first set of experiments we investigated whether the PreS fusion protein 4xP, which contains four copies of the B cell epitope–containing peptide P, can be used to induce a peptide P–specific IgE response in BALB/c mice. f 68.56% . No diffncreased . Similar3-adsorbed 2xP and boosted with 2xP did not develop any detectable peptide-specific IgE or IgG1 response response , 5E.Next we performed basophil activation experiments to determine whether 4xP-induced IgE Abs induce cross-linking of basophil-bound IgE and degranulation. For this purpose, RBL cells were loaded with serum IgE obtained from 4xP-sensitized and -boosted mice  on day 11 responses, the groups of mice were boosted on day 165, when we also observed a decline of peptide P–specific IgG1 Ab levels (p = 0.04) and 4xP (p = 0.04)   whereas  = 0.04) . As the  = 0.04) , group CNext, we investigated whether changes of peptide-specific IgE from day 164 to day 172 would have effects on basophil degranulation. For this purpose, pooled serum samples obtained at days 164, 172, and 179 from groups A–F were loaded on RBL-2H3 cells and degranulation was induced by a peptide–OVA conjugate, in which the peptide (P) was coupled to a carrier (OVA) with which the mice had not been in contact. The strongest increase of degranulation with the peptide–OVA conjugate was observed when basophils were loaded with sera from oligo-P–boosted mice from group C  , whereas1 Abs in the same groups of mice that had received a single immunization with 4xP  (1 Ab levels (p < 0.05)  , 8. Simiization) , oligo-P < 0.05) , group Cesponses . InteresE levels , 8. Miceese mice , group Fificance , group D1 Ab responses.These results illustrate that oligo-P, which contained a different carrier molecule than that used for sensitization and therefore did not receive T cell help by carrier-specific T cells, can boost peptide-specific IgE and IgG1 Ab responses. Spleen cells were isolated from the groups of mice that were immunized with 4xP and then boosted with the different Ags on day 185 , whereas peptide-specific IgG1 levels remained high for a longer period (after day 42). The earlier rise of IgE might be explained by a direct class switch to IgE, which was hypothesized earlier  (data not shown), we think that the increase of peptide-specific IgE response is not due to an induction of another “primary” IgE response but results from a direct stimulation of B memory cells by the oligomeric construct via cross-linking of the BCR. There are several possibilities for cross-linking of BCRs by allergens. First, it is possible that allergens contain repetitive IgE epitopes (Thus, immunization of mice with 4xP allowed the induction of a peptide-specific and thus allergen-specific IgE and IgG earlier , 36, whewith IgE  and/or dwith IgE , 39 thanwith IgE . We thenwith IgE . Howeverepitopes . Second,epitopes , 45. Thiepitopes , 47. T cepitopes , 23 or tepitopes . So far epitopes –19. HoweWe think that our findings are also important for the design of new forms of SIT, because it may be considered to use recombinant allergen derivatives that primarily induce allergen-specific IgG responses without activating allergen-specific T cell epitopes , 47–50 fData Supplement


Deficits in GABAergic inhibitory neurotransmission are a reliable finding in schizophrenia (SCZ) patients. Previous studies have reported that unaffected first-degree relatives of patients with SCZ demonstrate neurophysiological abnormalities that are intermediate between probands and healthy controls. In this study, first-degree relatives of patients with SCZ and their related probands were investigated to assess frontal cortical inhibition. Long-interval cortical inhibition (LICI) was measured from the dorsolateral prefrontal cortex (DLPFC) using combined transcranial magnetic stimulation (TMS) and electroencephalography (EEG). The study presents an extended sample of 129 subjects (66 subjects have been previously reported): 19 patients with SCZ or schizoaffective disorder, 30 unaffected first-degree relatives of these SCZ patients, 13 obsessive-compulsive disorder (OCD) patients, 18 unaffected first-degree relatives of these OCD patients and 49 healthy subjects. In the DLPFC, cortical inhibition was significantly decreased in patients with SCZ compared to healthy subjects. First-degree relatives of patients with SCZ showed significantly more cortical inhibition than their SCZ probands. No differences were demonstrated between first-degree relatives of SCZ patients and healthy subjects. Taken together, these findings show that more studies are needed to establish an objective biological marker for potential diagnostic usage in severe psychiatric disorders. SCZ is a severe psychotic disorder characterized by positive symptoms, negative symptoms and cognitive impairments123In psychiatry, there are no objective laboratory tests to inform diagnoses and monitor response to treatment. Endophenotypes are genetically determined phenotypes that may be part of a complex disease and facilitate the development of etiologic rather than symptom-based diagnostic methods. They help to advance our understanding of the genetic mechanisms underlying psychiatric disorders6et al. reported a decreased density of non-pyramidal cells in anterior cingulate layers II-VI and in prefrontal cortex layer II in SCZet al. found reduced messenger RNA (involved in the synthesis of GABA) in the dorsolateral prefrontal cortex (DLPFC) of SCZ patients1213141516171819et al.Deficits in GABAergic inhibitory neurotransmission have been a reliable finding in SCZ across multi-modal investigative approaches. These deficits may be due to an imbalance between cortical excitation and inhibition of the cortexB receptor-mediated inhibitory neurophysiological mechanisms can be measured through a paired-pulse paradigm, long-interval cortical inhibition (LICI). Farzan et al. have demonstrated using TMS-EEG that LICI of gamma oscillations were selectively impaired in the DLPFC of patients with SCZ compared to both healthy subjects and similarly treated patients with bipolar disorderTMS combined with electroencephalography (EEG) is a powerful tool for investigating cortical mechanisms and networks of frontal brain areas. Recent technical advances have enabled the concurrent recording of TMS and EEG. Using this approach, GABAB-mediated cortical inhibition in patients with SCZ, patients with OCD patients, both of their unaffected first-degree relatives compared to healthy subjects. Based on previous studies22The objective of the present study was to evaluate frontal GABAThe study assessed 129 subjects, The Yale-Brown Obsessive Compulsive Scale (YBOCS) was used for evaluating psychopathology in OCD patients27Monophasic TMS pulses were administered using a 7 cm figure-of-eight coil, and two Magstim 200 stimulators  connected via a Bistim module. TMS was administered over the left motor cortex and DLPFC in separate blocks. One hundred TMS stimuli were delivered per-condition (paired and single-pulse) every 5 seconds. LICI was obtained at the 100 ms interstimulus intervalThe TMS coil was placed at the optimal position for eliciting motor evoked potentials from the right abductor pollicis brevis muscle, which corresponded to electrodes FC3 and C3. Electromyography was captured by placing two disposable disc electrodes over the right abductor pollicis brevis muscle and motor evoked potentials were filtered (band-pass 2 to 5 kHz), digitized at 5 kHz .Localization of DLPFC was achieved through neuronavigation techniques using the MINIBIRD system (Ascension Technologies) and MRIcro/registration software using a T1-weighted MRI scan obtained for each subject with seven fiducial markers in place22To evaluate TMS-induced cortical evoked potentials, EEG was recorded concurrently with electromyography. EEG was acquired through a 64-channel Synamps 2 EEG system. A 64-channel EEG cap was used to record the cortical signals, and four electrodes were placed on the outer side of each eye, and above and below the left eye to closely monitor eye movement artifacts. All electrodes were referenced to an electrode positioned posterior to Cz electrode. EEG signals were recorded DC and with a low pass filter of 100 Hz at a 20 kHz sampling rate, to avoid saturation of the amplifiers and to minimize TMS-related artifact31EEG recordings were down-sampled to 1000 Hz and epoched from −1000 ms to 2000 ms after the test TMS pulse. In both, the single and paired-pulse conditions, the data from −100 ms to 10 ms was fully removed (where 0 correspond to the test TMS pulse). This step removes the test-pulse TMS from both of the single-pulse and paired-pulse conditions and also the conditioning TMS pulse from the paired-pulse condition. Traces were visually inspected for artifacts in order to eliminate trials and channels highly contaminated by noise . Two rounds of independent component analysis were subsequently applied. The first round was to minimize and remove the typical TMS-related decay artifact. Following this, a bandpass FIR filter was applied from 1 to 55 Hz and a second round of independent component analysis was computed to remove eye movement-related artifacts (blinks and movements) and muscle components.2/Hz) after subtracting the log baseline to the whole trial. In two previous studies, we have shown that inhibition can be evaluated as the difference between the single and paired-pulse conditions, using a measure of amplitude of the evoked activity23Time-frequency decomposition was obtained using the Event-Related Spectral Perturbation (ERSP) analysis in EEGLab. Specifically the analysis was wavelet based, using a cycle of the complex Morlet wavelet across frequencies 2–50 Hz. The ERSP was computed independently for the single-pulse and paired-pulse conditions. The analysis is expressed in decibels of spectral power  was chosen to label each voxel as inhibited = 1 or not-inhibited = 0. A voxel received a value of “1” if its value is greater than 99% of the values in the same voxel in the null distribution, otherwise the value was set at “0”. The number of randomization in the null distribution was set to 500. Inhibition was then evaluated by counting the 1’s that forms a cluster in this time-frequency-electrode domain. A voxel belongs to a cluster if it has a value of 1, and has at least a neighbour in time, frequency and electrode that is also in the cluster23After calculating inhibition by subject, we compared groups by a t-test with pooled variance. In all comparisons, the two-tailed analyses are reported. However, based on our previous finding23DLPFC inhibition  was compared between young (<50 years) first-degree relatives of SCZ patients and older (>50 years) first-degree relatives of SCZ patients to determine whether there were effects of age. An independent samples t-test was used.A Spearman’s rho correlation analysis was performed between the SPQ and the index of frontal inhibition  for the first-degree relatives of SCZ group.A Spearman’s rho correlation analysis was performed between the BPRS and the index of frontal inhibition  for each SCZ patients. A Spearman’s rho correlation analysis was performed between the YBOCS and the index of frontal inhibition  for each OCD patient. A Spearman’s rho was also conducted with antipsychotic medication dosage expressed as chlorpromazine equivalents343536DLPFC inhibition  was compared with antidepressant-treated patients and patients not treated with antidepressants in both SCZ and OCD patient groups. An independent samples t-test was used. DLPFC inhibition  was compared with benzodiazepine-treated patients and patients who were not treated with benzodiazepines in both SCZ and OCD patient groups. An independent samples t-test was used.Variance components methods in SOLAR v.4.3.1 were used to estimate the narrow sense heritability for overall inhibition of the DLPFC, defined as the phenotypic variance explained by additive genetic factorsFrontal inhibition was significantly greater in healthy subjects compared to subjects with SCZ . First-degree relatives of SCZ patients showed significantly more inhibition than their SCZ probands . No differences were demonstrated between first-degree relatives of SCZ and healthy subjects . Gamma inhibition was significantly lower in SCZ compared to healthy subjects . No significant differences were found between healthy subjects and first-degree relatives of SCZ . When comparing first-degree relatives of SCZ to their probands, non-significant differences in gamma inhibition were shown . No significant differences were found between healthy controls compared to OCD  and when compared to first-degree relatives of OCD . Lastly, no significant differences were found between first-degree relatives of OCD compared to their OCD probands .No significant inhibitory differences were found between healthy subjects and SCZ patients  or healthy subjects as compared to their first-degree relatives . No significant differences were found between SCZ patients and their first-degree relatives . No differences were found between healthy subjects compared to OCD patients  and when compared to their first-degree relatives . No significant differences were found between OCD patients and their first-degree relatives .No significant inhibitory differences were found between healthy subjects and SCZ patients  or healthy subjects as compared to their first-degree relatives . No significant differences were found between SCZ patients and their first-degree relatives . No differences were found between healthy subjects compared to OCD patients  and when compared to their first-degree relatives . No significant differences were found between OCD patients and their first-degree relatives .No significant differences were found when comparing younger (n = 9) (<50 years) to older (n = 21) (>50 years) first-degree relatives of SCZ patients in frontal overall inhibition (p = 0.48) and frontal gamma inhibition (p = 0.66).In first-degree relatives of SCZ patients, no significant relationship was found between the SPQ  and frontal overall inhibition . In first-degree relatives of SCZ, no significant relationship was found between the SPQ  and frontal gamma inhibition .No significant correlation between overall inhibition and chlorpromazine equivalents was shown  and no relationship was found between frontal gamma inhibition and chlorpromazine equivalents .In the DLPFC, no significant differences were found between antidepressant-treated OCD patients (n = 7) and unmedicated OCD patients (n = 6) in overall inhibition (p = 0.15) and gamma inhibition (p = 0.32). In the DLPFC, no significant differences were found between antidepressant-treated SCZ patients (n = 4) and SCZ patients who were not treated with antidepressants (n = 15) in overall frontal inhibition (p = 0.89) and frontal gamma inhibition (p = 0.92). In the DLPFC, no significant differences were found between OCD patients who were treated with SSRIs (n = 4) and SCZ patients who were treated with SSRIs (n = 2) in overall frontal inhibition (p = 0.40) and gamma inhibition (p = 0.31).Lastly, in the DLPFC, no significant differences were found between benzodiazepine-treated OCD patients (n = 4) and OCD patients (n = 9) who were not treated with benzodiazepines in overall frontal inhibition (p = 0.78) and frontal gamma inhibition (p = 0.95). We also found no significant differences between benzodiazepine-treated SCZ patients (n = 4) and SCZ patients (n = 15) who were not treated with benzodiazepines in overall frontal inhibition (p = 0.31) and frontal gamma inhibition (p = 0.66).No significant relationship was found between the BPRS and overall frontal inhibition, . No significant relationship was found between the BPRS and frontal gamma inhibition .No significant relationship was found between the YBOCS and overall frontal inhibition, . No significant relationship was found between the YBOCS and frontal gamma inhibition .The receiver operating characteristic (ROC) curve analysis was completed to quantify the cluster size for both overall inhibition and gamma inhibition as a classifier based on the sensitivity and specificity. Healthy and OCD patients were combined to compare to SCZ. The reported values were the area under the ROC curve indexing a combined sensitivity and specificity. A significant area greater than 0.50 indicates that the value is better than a guess at random (i.e. flipping a coin) and a perfect test is 1.0. Youden’s J statistic was reported and demonstrates the peak of the ROC curve to index specificity and sensitivity seperatelyWe found that first-degree relatives of SCZ patients showed an intermediate pattern of frontal inhibition compared to their related probands and healthy controls. Significant differences were found in frontal overall inhibition between SCZ patients and their unaffected first-degree relatives. Significant deficits were shown in frontal overall and gamma inhibition in patients with SCZ compared to healthy subjects. A sensitivity of 89% for frontal gamma inhibition was found.We demonstrated that first-degree relatives of SCZ had significant differences in overall inhibition when compared to their related probands. First-degree relatives of SCZ were not significantly different from healthy subjects. The pattern of frontal inhibition in first-degree relatives of SCZ was intermediate between their related probands and healthy controls .et al. demonstrated that P50 suppression shows a low heritability of 0.10 that was not significant in a sample of 183 nuclear familieset al. demonstrated moderate heritability of 0.45 for prepulse inhibition at the 60 ms interstimulus interval and a trending heritability of 0.33 at the 120 ms interstimulus intervalData from biological relatives of probands are important for assessing disease-related effects in a complex disorder like SCZ. The Consortium of Genetics in Schizophrenia (COGS) has investigated several neurophysiological measures as potential endophenotypes as a means for understanding the genetic determinants of SCZCurrently, no objective measures exist to inform psychiatric diagnoses as the clinical interview dominates the diagnostic approach. Psychiatric disorders are complex, no specific constellation of genes or environmental conditions characterize a large subset of ill individuals44In this study, we found a sensitivity of 89% for gamma inhibition, also known as the true-positive rate. This finding suggests that gamma inhibition can accurately identify SCZ at a rate of 89%, providing a positive test result. Gamma inhibition has been shown to be a more informative measure based on the classification analyses. Gamma inhibition of the DLPFC may be explored further in SCZ based on the high sensitivity results.GABAergic deficits have been found in SCZ based on post-mortem and animal studies showing reduced expression of pre- and postsynaptic markers of GABAergic neurotransmission in subpopulations of GABAergic interneurons in the prefrontal cortex474851525355The main challenge of family-based studies is recruitment. Larger samples of first-degree relatives are needed to increase power and address the heritability question. In future, multi-center research trials are needed to develop TMS-EEG as a neurophysiological method for use in clinical settings. Furthermore, patients with SCZ were treated with a variety of antipsychotic medications and other psychotropic medications and were chronically ill, which may have effects on neural oscillations. Future studies should recruit unmedicated patients to assess these medication effects.This study shows that impairments in frontal inhibition are specific to the pathophysiology of SCZ and may have the potential for use in diagnosis. The search for liability genes for complex disorders such as SCZ may be aided by identifying endophenotypes and relating these genes to cortical inhibition.How to cite this article: Radhu, N. et al. Investigating Cortical Inhibition in First-Degree Relatives and Probands in Schizophrenia. Sci. Rep.7, 43629; doi: 10.1038/srep43629 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Likewise, Thr383 phosphorylation is involved in β-arrestin-dependent Erk1/2 stimulation elicited by other GPCRs such as β2-adrenergic, FSH and CXCR4 receptors, but does not affect the β-arrestin-independent Erk1/2 activation by 5-HT4 receptor. Collectively, these data show that β-arrestin2 phosphorylation at Thr383 underlies β-arrestin-dependent Erk1/2 activation by GPCRs.In addition to their role in desensitization and internalization of G protein-coupled receptors (GPCRs), β-arrestins are essential scaffolds linking GPCRs to Erk1/2 signaling. However, their role in GPCR-operated Erk1/2 activation differs between GPCRs and the underlying mechanism remains poorly characterized. Here, we show that activation of serotonin 5-HTDOI:http://dx.doi.org/10.7554/eLife.23777.001 Over the last decade, it has become evident that β-arrestins are also important signal transducers and that a number of biological functions exerted by GPCRs are mediated by β-arrestin-dependent signaling . β-arresrrestins , whereasruitment . In addiruitment .412 for β-arrestin1  was identified in rat/mouse β-arrestin2 and its phosphorylation state affects endosomal trafficking of various GPCRs  . This phrrestin1  and Thr2rrestin2 . These preceptor , 2002, wreceptor . These rus GPCRs . Howeverus GPCRs . In cont2C and 5-HT4 receptors, two GPCRs that differ in their β-arrestin dependency to promote Erk1/2 activation, using high-resolution mass spectrometry. We identified several previously described as well as novel phosphorylated residues on β-arrestin2, while only one phosphorylated site was found on β-arrestin1. Of these, only phosphorylation of β-arrestin2 at Thr383 exhibited a strong increase upon 5-HT2C receptor stimulation. Furthermore, Thr383 was poorly phosphorylated in cells expressing 5-HT4 receptor and its phosphorylation was only slightly enhanced by agonist treatment. These findings prompted functional studies to evaluate the influence of this phosphorylation event in Erk1/2 phosphorylation elicited by stimulation of 5-HT2C receptor and other GPCRs known to engage Erk signaling in a β-arrestin-dependent manner, in comparison with 5-HT4 receptor stimulation, which induces Erk1/2 activation through a β-arrestin-independent mechanism.Here, we investigated the impact on β-arrestin1 and 2 phosphorylation of expression/stimulation of 5-HT2C and 5-HT4 receptor expression/stimulation on the phosphorylation state of β-arrestins, YFP-tagged versions of β-arrestin1 or β-arrestin2 were co-expressed with Myc-tagged 5-HT2C or 5-HT4 receptors in HEK-293 cells. Cells were then treated with vehicle or 5-HT for 5 or 30 min. β-arrestins were immunoprecipitated using GFP nanobodies coupled to sepharose beads (GFP-Trap), resolved by SDS-PAGE, detected by colloidal Coomassie blue staining  was identified on β-arrestin1, but the corresponding phosphorylated peptide was only detected in cells expressing 5-HT4 receptor, and with a low phosphorylation index, as estimated by dividing the MS signal intensity of the phosphorylated peptide by the sum of MS signal intensities of the phosphorylated and the corresponding non-phosphorylated peptide  were identified on β-arrestin2 (267S268)TFCK peptide did not provide enough information to discriminate between a phosphorylation at Ser267 or at Ser268, but the precursor peptide m/z clearly showed that it carries only one phosphorylated residue , consistent with previous findings . Numerous studies have shown that β-arrestins associated with a GPCR can interact with different signaling proteins including c-Src and several proteins of the Erk signaling module (Raf/MEK/Erk), and that these interactions depend on β-arrestin conformational state  the receptor C-terminus associates with β-arrestin2 using the region previously occupied by the 383–393 β-strand within the β-arrestin2 C-terminal tail and (iv) Erk binds to the complex and can be activated by MEK  docking algorithm, and validated experimentally the previously unknown interaction regions . This moive site , indicatd by MEK . Consistmulation . Expressrylation . The abirylation . Collectn medium . As expein vitro .383 phosphorylation affects the recruitment of β-arrestin2 by 5-HT2C and 5-HT4 receptors, we monitored interaction between each receptor and either wild type or Thr383Ala or Thr383Asp β-arrestin2 mutants using a BRET-based assay. Exposure of cells to 5-HT induced a strong increase in the BRET signal between 5-HT2C-YFP or 5-HT4-YFP receptor and Rluc-β-arrestin2  and the Renilla luciferase variant Rluc8  receptor, β-adrenergic receptors and chemokine CXCR4 receptor  and three new sites . Two of the β-arrestin2 phosphorylated sites identified  are conserved in β-arrestin1, but we did not detect the corresponding phosphorylated peptides in β-arrestin1. Although MS/MS does not allow exhaustive identification of peptides present in complex samples, it is unlikely that our analyses, which covered about of 85% of the sequence of both arrestins, missed these phosphorylated residues on β-arrestin1 or any other robustly phosphorylated residues on both arrestins. Therefore, our results corroborate previous findings which identified much more phosphorylated residues on β-arrestin2 than on β-arrestin1 and suggest that β-arrestin2 is phosphorylated at higher stoichiometry, compared with β-arrestin1. Our analyses did not detect, even in 5-HT-treated cells, another site conserved between both β-arrestins (Ser276) that was previously reported as being phosphorylated by recombinant active Erk1 in an in vitro phosphorylation assay followed by MS/MS analysis    and β-arDB 1G4R) , the segDB 1G4R) , also shDB 1G4R) . Finallyur model  and 6, T2C receptor, a GPCR which elicits Erk1/2 signaling mainly through a β-arrestin-dependent mechanism, appears to be conserved among GPCRs that induce Erk1/2 activation through the classic dual mechanism involving both G proteins and β-arrestins, such as β-adrenergic, CXCR4 and FSH receptors. Actually, the sustained Erk1/2 phosphorylation induced by agonist stimulation of these receptors was strongly reduced in cells expressing the Thr383Ala β-arrestin2 mutant. In contrast, substitution of Thr383 by aspartate increased the basal level of Erk1/2 phosphorylation, suggesting that phosphorylated β-arrestin2 can recruit and subsequently activate Erk1/2 in absence of receptor stimulation. As expected, 5-HT4 receptor-operated Erk1/2 phosphorylation, which is β-arrestin-independent, was not affected by substituting Thr383 by alanine. This result is consistent with the low Thr383 phosphorylation level measured in cells expressing 5-HT4 receptors, compared with cells expressing 5-HT2C, and suggests that Thr383 phosphorylation might be a key molecular event governing β-arrestin dependency of Erk1/2 pathway engagement by GPCRs.This mechanism, initially established for the 5-HT383 as a key molecular event underlying β-arrestin-dependent Erk1/2 activation by GPCRs. Thr383 phosphorylation has recently been involved in β-arrestin2 association with transient receptor potential vanilloid 1 (TRPV1) at the plasma membrane, which is critical for its role in regulating receptor desensitization  might govern steady state level of GPCR cell-surface expression  were from the American Type Culture Collection . All experiments of the study were performed using a previously characterized batch of cells (Isolate #3) . The siRNA used to target β-arrestin2  and the control siRNA (5’-AAUUCUCCGAACGUGUCACGU-3’) were from Dharmacon and the GeneSilencer siRNA transfection reagent from Genlantis .Human Embryonic Kidney-293 (HEK-293) N type cells (RRID:late #3) . Their nT383A and pcDNA3-Rluc-βARR2T383D constructs were generated by site directed mutagenesis from the pcDNA3-Rluc-βARR2 vector. The pCMV-Rluc8-βARR2-RGFP construct used to monitor β-arrestin2 conformational changes (double brilliance assays), previously described in (T383A-RGFP and pCMV-Rluc8-βARR2T383D-RGFP were generated by site directed mutagenesis from this vector. Plasmids encoding Myc-5-HT2C-YFP receptor (pEYFP-N1-Myc-5-HT2C) and Myc-5-HT4-YFP receptor (pEYFP-N1-Myc-5-HT4) were generated from pRK5-Myc-5-HT2C-VGV and pRK5-Myc-5-HT4 plasmids . pcDNA3-Rluc-βARR2ribed in , was a gplasmids , respectplasmids . The pla202/Tyr204-Erk1/2 antibody , the rabbit anti-GAPDH antibody (RRID:AB_10167668) and the goat anti-integrin β1 antibody (RRID:AB_2128200) were from Santa Cruz Biotechnology  , the rabbit anti-pan arrestin antibody (RRID:AB_303409) and the mouse anti-β-arrestin2 antibody (RRID:AB_2060273) from Abcam (UK), the rabbit anti-Erk1/2 antibody (RRID:AB_330744), the rabbit anti-Elk1 (RRID:AB_2277936) and the rabbit anti-phospho-Ser383-Elk1 (RRID:AB_2099016) from Cell Signaling Technology , the rabbit anti-GFP antibody (RRID:AB_221569) from Invitrogen, and the rabbit anti-Flag (RRID:AB_439687) and mouse anti-Myc (RRID:AB_439694) antibodies from Sigma Aldrich . The anti-phosphoThr383- β-arrestin2 antibody was generated by immunizing rabbits with the synthetic CDTNYAT(PO3H2)DDDIVF peptide coupled to Keyhole Limpet Hemocyanin . Antibodies were purified by two consecutive affinity chromatographies. A first chromatography against the immobilized phosphorylated CDTNYAT(PO3H2)DDDIVF peptide as bait bound antibodies recognizing both phosphorylated and non-phosphorylated forms of the peptide. The retained fraction was then eluted and used for a second chromatography against the immobilized non-phosphorylated CDTNYATDDDIVF peptide as bait. We considered that the non-retained fraction contains antibodies recognizing β-arrestin2 phosphorylated at Thr383. 5-HT, isoproterenol and human recombinant FSH were from Sigma Aldrich, U0126 from Promega , FR180204 and TBCA from Tocris Bioscience (UK) and CXCL12 from R&D Systems . [γ-32P]-ATP was from Perkin Elmer .The mouse anti-phospho-Thr3H]-mesulergine and [3H]-GR113808 binding experiments showed that 5-HT2C and 5-HT4 receptor densities in transfected HEK-293 cells were 0.29 and 0.90 pmol/mg protein, respectively. Based on previously published levels of the endogenous β-arrestins in HEK-293 cells (6 cells) and plasmids encoding receptors of interest, using the GeneSilencer reagent.HEK-293 cells, grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% dialyzed, heat-inactivated fetal calf serum and antibiotics, were transfected at 40–50% confluence using polyethyleneimine , as previously described  and used93 cells , we also2C or Myc-5-HT4 receptor were starved of serum for 4 hr, challenged with either vehicle or 5-HT and lysed in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP40, 0.4% dodecylmaltoside, 10 mM sodium fluoride, 1 mM sodium pyrophosphate, 50 mM β-glycerophosphate, 2 mM sodium orthovanadate and a protease inhibitor cocktail . Samples were centrifuged at 15,000 × g for 15 min at 4°C and β-arrestins were immunoprecipitated with agarose-conjugated GFP trap . After reduction with dithiothreitol  and alkylation with iodoacetamide , immunoprecipitated β-arrestins were resolved by SDS-PAGE and digested in-gel overnight at 25°C with trypsin . Peptides were analyzed by nano-LC-FT-MS/MS using a LTQ Orbitrap Velos mass spectrometer coupled to an Ultimate 3000 HPLC . Desalting and pre-concentration of samples were performed on-line on a Pepmap precolumn . A gradient consisting of 2–40% buffer B (3–33 min), 40–80% B (33–34 min), 80–0% B (49–50 min), and equilibrated for 20 min in 0% B (50–70 min) was used to elute peptides at 300 nL/min from a Pepmap capillary (0.075 mm × 150 mm) reversed-phase column (Thermo Fisher Scientific). Mass spectra were acquired using a top-10 collision-induced dissociation (CID) data-dependent acquisition (DDA) method. The LTQ-Orbitrap was programmed to perform a Fourier transform (FT) full scan  on 400–1,400 Th mass range with the top ten ions from each scan selected for LTQ-MS/MS with multistage activation on the neutral loss of 24.49, 32.66 and 48.99 Th. FT spectra were internally calibrated using a single lock mass (445.1200 Th). Target ion numbers were 500,000 for FT full scan on the Orbitrap and 10,000 MSn on the LTQ. Precursor mass and top 6 per 30 Da windows peak lists were extracted from MS2 using MSconvert 3.0 and searched with Mascot 2.4 (RRID:SCR_014322) against the same human Complete Proteome Set database (RRID:SCR_002380), cysteine carbamidomethylation as a fixed modification and phosphorylation of Ser, Thr and Tyr as variable modifications, 7 ppm precursor mass tolerance, 0.5 Da fragment mass tolerance and trypsin/P digestion. MS2 spectra matching phosphorylated peptides with ion score over 15 were manually inspected for unique transitions that pinpoint the position of phosphorylation sites. Ion signals corresponding to phosphorylated peptides were quantified from the maximal intensities measured in their ion chromatograms manually extracted using Qual browser v2.1 (Thermo Fisher Scientific) with a tolerance of 5 ppm for mass deviation, and normalized to signals of their non-phosphorylated counterparts. For each identified phosphorylated residue, a phosphorylation index  was calculated.HEK-293 cells transiently co-expressing YFP-tagged β-arrestin1 or YFP-tagged β-arrestin2 and either Myc-5-HT383Ala mutant) was purified from HEK-293 cells using GFP trap beads, as described in the previous section. For kinase assay, 10 µL of purified fractions containing ~1 µg β-arrestin2 were incubated for 10 min at 37°C in 5 µL kinase buffer  in absence or presence of 5 µL active MEK1 . Kinase reaction was started by adding 5 µL ATP (250 µM) and was stopped by adding Laemmli buffer. β-arrestin2 phosphorylation at Thr383 was analyzed by Western blotting using the purified anti-phospho-Thr383-β-arrestin2 antibody. For radioactive kinase assay, 10 µl of purified fractions containing ~1 µg YFP-β-arrestin2 or 1 µg of non-activated, purified GST-tagged Erk2 (Sigma Aldrich) were incubated for 10 min at 37°C in 5 µL kinase buffer in presence of active MEK1 and [γ-32P]-ATP . Proteins were separated by SDS-PAGE and stained with colloidal Coomassie. Gels were dried and exposed for autoradiography. Radioactive bands corresponding to β-arrestin2 and Erk2 were excised and radioactivity was measured by β-scintillation counting.YFP-tagged β-arrestin2  and then with either anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies . Immunoreactivity was detected with an enhanced chemiluminescence method . Immunoreactive bands were quantified by densitometry using the ImageJ software. In protein phosphorylation analyses, the amount of each phosphoprotein was normalized to the corresponding total protein signal.Proteins, resolved onto 10% polyacrylamide gels, were transferred to Hybond C nitrocellulose membranes . Membranes were immunoblotted with primary antibodies (anti phospho-Thr2C and 5-HT4 receptors was performed by ELISA under permeabilized (Triton X-100 0.05%) vs. non-permeabilized conditions, as previously described  for 48 hr and starved of serum for 6 hr. Cells were washed once with PBS and Coelenterazine H (Sigma-Aldrich) was added at a final concentration of 5 μM for 10 min. Cells were then challenged with 5-HT for 30 min. BRET readings were performed using a Mithras LB940 multimode microplate reader  allowing sequential integration of luminescence with three filter settings: Rluc filter, 485 ± 10 nm; YFP filter, 530 ± 12.5 nm; RGFP filter, 515 ± 10 nm. Emission signals at 530 nm or 515 nm were divided by emission signals at 485 nm. The difference between this emission ratio obtained with donor and acceptor co-expressed or fused and that obtained with the donor protein expressed alone was defined as the BRET ratio. Results are expressed in milliBRET units corresponding to the BRET ratio multiplied by 1,000.HEK-293 cells were transiently co-transfected with either pEYFP-N1-Myc-5-HTModeling of the receptor/ß-arrestin/Erk module was made using the PRIOR method, and the PDB structures and methodology indicated in . StructuStatistical analyses were performed using Prism . Unpaired t test and one-way ANOVA followed by Newman Keuls test were performed to compare two values and for multiple comparisons, respectively.For each graph, the number of biological replicates (independent experiments performed on different sets of cultured cells) and significance of observed differences are indicated in the figure legend. The different levels of statistical significance are encoded with symbols  directly on the histograms and explained in the figure legends. The statistical tests and software used are indicated in 'Materials and methods' section.MS peptide identification probabilities were performed using the Mascot algorithm. Mascot search was performed using parameters described in the 'Material and methods' section. Mascot score represents −10*LOG10, where p-value is the absolute probability of peptide wrong assignment. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.383 by MEK underlies β-arrestin-dependent activation of Erk1/2 by GPCRs" for consideration by eLife. Your article has been favorably evaluated by Tony Hunter (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. Our decision has been reached after consultation between the reviewers. Based on these discussions, we regret to inform you that we cannot accept your manuscript for publication in eLife. We are therefore returning the manuscript to you at this time. Substantial revision will be required to address the criticisms raised during the review. If you are able to fully address these criticisms after the completion of new experimentation, we would happy to consider a new manuscript for publication in eLife.Thank you for submitting your work entitled "Phosphorylation of β-arrestin2 at ThrSummary:2C receptors activate the ERK pathway via the β-arrestin2 scaffold protein. The authors propose that MEK promotes the phosphorylation of β-arrestin2 at Thr383 and that this is necessary for recruitment of ERK to the receptor complex and its subsequent activation. The findings are potentially very interesting and reveal a novel signaling mechanism for the activation of ERK MAPK downstream of GPCRs. However, further experiments are required to confirm the proposed model and it is a concern that all the experiments rely on the ectopic expression of β-arrestin2 and GPCRs in cells.This study reports the mechanism by which serotonin 5-HTMajor Points:383 on β-arrestin2. Does purified MEK phosphorylate β-arrestin2 in vitro? How does MEK phosphorylation of β-arrestin2 compare with that of known MEK substrates?1) There is no direct evidence presented that MEK phosphorylates Thr383 phosphorylation of endogenous β-arrestin2 to regulate ERK signaling.2) All the experiments, including the MS analysis, are performed on overexpressed YFP-β-arrestin2. It is important to demonstrate that 5-HT induces Thr3) The proposed model is that MEK phosphorylation of β-arrestin2 promotes ERK recruitment. To support this, it should be shown that MEK inhibition prevents ERK recruitment to β-arrestin2.383 on β-arrestin2. The authors suggest in the Discussion that CK2 may be responsible for the basal levels of Thr383 phosphorylation. They should demonstrate that inhibition of CK2 does not affect 5-HT-induced Thr383 phosphorylation.4) CK2 has previously been reported to phosphorylate Thr383 phosphorylation causes recruitment of ERK nor do the data presented inform us about the activation mechanism, since increased ERK retention could equally well result in a block in ERK activation.5) It is unclear how Thr6) The relative expression of proteins (e.g. receptor and scaffold) in the transfection assays is unclear, and needs to be included.383 phosphorylation (7) The authors could have provided a more coherent description of the proposed model. The BRET data suggests that the N and C-termini of β-arrestin2 come into closer proximity upon 5-HT treatment and that this is promoted by Thrrylation . This is[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]383 by MEK underlies β-arrestin dependent activation of Erk1/2 by GPCRs" for further consideration at eLife. Your revised article has been favorably evaluated by Tony Hunter (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Phosphorylation of β-arrestin2 at ThrThe manuscript has been substantially improved, but there is one remaining issue that needs to be addressed before acceptance, as outlined below:383 in β-arrestin2 in vitro are important. However, these data do not address the question in the original decision: "How does MEK phosphorylation of β-arrestin2 compare with that of a known MEK substrate?" A direct comparison of the efficiency of phosphorylation of β-arrestin2 and ERK using an in vitro kinase assay is needed.Point 1: The new data demonstrating that MEK phosphorylates Thr [Editors’ note: the author responses to the first round of peer review follow.]Major Points:383 on β-arrestin2. Does purified MEK phosphorylate β-arrestin21) There is no direct evidence presented that MEK phosphorylates Thr in vitro? How does MEK phosphorylation of β-arrestin2 compare with that of known MEK substrates?383 (see Point 2), we show that purified MEK phosphorylates β-arrestin2 purified from HEK-293 cells on Thr383 . As expected, no increase in Thr383 phosphorylation was seen in presence of the MEK inhibitor U0126.Using an in vitro kinase assay followed by Western blotting performed with a newly generated polyclonal antibody against phosphorylated Thr383 phosphorylation of endogenous β-arrestin2 to regulate ERK signaling.2) All the experiments, including the MS analysis, are performed on overexpressed YFP-β-arrestin2. It is important to demonstrate that 5-HT induces Thr383 (see on 383) and showed that 5-HT2C receptor stimulation, induces a substantial increase in endogenous β-arrestin2 on Thr383, while activation of the 5- HT4 receptor (known to activate Erk1/2 via a β-arrestin-independent mechanism) does not affect its phosphorylation state, corroborating the results of MS/MS analysis of ectopic β- arrestin2 phosphorylation .We agree with the reviewers that it is an important issue. Unfortunately, we did not detect by MS/MS any phosphorylated peptide of endogenously expressed arrestins, even after their immunoprecipitation from large cell lysate amounts, likely due to their low expression level in HEK-293 cells and the low immunoprecipitation yield (less than 10%) using commercially available anti-β-arrestin2 antibodies. We thus generated and validated a polyclonal antibody against phosphorylated Thr3) The proposed model is that MEK phosphorylation of β-arrestin2 promotes ERK recruitment. To support this, it should be shown that MEK inhibition prevents ERK recruitment to β-arrestin2.2C receptor expressing cells with the MEK inhibitor U0126 abolishes Erk recruitment to β-arrestin2 (assessed by co- immunoprecipitation). This result is illustrated on 383 is essential for Erk1/2 translocation to the 5-HT2C receptor/β-arrestin2 complex”).Consistent with the proposed model, we show that treating 5-HT383 on β-arrestin2. The authors suggest in the Discussion that CK2 may be responsible for the basal levels of Thr383 phosphorylation. They should demonstrate that inhibition of CK2 does not affect 5-HT-induced Thr383 phosphorylation.4) CK2 has previously been reported to phosphorylate Thr383 phosphorylation elicited upon 5-HT2C receptor stimulation is not affected by treating cells with the selective cell-permeable CK2 pharmacological inhibitor tetrabromocinnamic acid . Note that previously published studies only reported the phosphorylation of this residue by purified CK2 in vitro. Our results suggest that CK2 plays a minor role (if any) in Thr383 phosphorylation in HEK-293 cells and that MEK might be the main kinase responsible for this phosphorylation in living cells. We have thus modified the Discussion accordingly (second paragraph).We show that Thr383 phosphorylation causes recruitment of ERK nor do the data presented inform us about the activation mechanism, since increased ERK retention could equally well result in a block in ERK activation.5) It is unclear how Thr383 by MEK, within the receptor/β-arrestin2/Raf/MEK complex, is responsible for a large conformational change of β-arrestin2 region comprising residues 350-393. This model is based on (i) the fact that this region in inactive β-arrestin2 interacts with the N-terminal region and has to be displaced for the interaction of this N-terminal region of β-arrestin with the receptor C-tail, (ii) our experimentally validated docking model which shows that in the receptor/β-arrestin/Raf/MEK/Erk complex, the receptor C-tail is sandwiched between β-arrestin and Erk and, consequently, that the conformational change has to take place before interaction with Erk and (iii) our experiments indicating that Erk is not recruited to the complex in absence of this phosphorylation. This is of course only a model. Though different hypotheses could be formulated, this model led us to make the hypothesis and then to validate that MEK is responsible for Thr383 phosphorylation, and it accounts for all the existing knowledge. We have largely modified the Discussion (fifth paragraph) to better distinguish the elements that are demonstrated and those which remain hypothetical.Numerous studies have demonstrated that recruitment of Erk, together with Raf and MEK, to activated GPCR/β-arrestin complex results in Erk activation. In this paper, we propose a mechanistic model whereby β-arrestin2 phosphorylation of Thr6) The relative expression of proteins (e.g. receptor and scaffold) in the transfection assays is unclear, and needs to be included.2C and 5-HT4 receptors  and an estimation (thanks to Western blot experiments) of cellular levels of β-arrestin1 and β-arrestin2 in transfected cells , based on previously published levels of the endogenous proteins in HEK-293 cells .We now provide in “Materials and methods” (subsection “Cell cultures and transfections”) the expression levels of 5-HT383 phosphorylation (7) The authors could have provided a more coherent description of the proposed model. The BRET data suggests that the N and C-termini of β-arrestin2 come into closer proximity upon 5-HT treatment and that this is promoted by Thrrylation . This is383 phosphorylation. Such experiments do not provide information regarding the exact nature of the conformational change. Our mechanistic hypotheses only concern the region of the β-arrestin2 C-tail comprising residues 350-393, and do not suggest conformational changes affecting the C-terminal extremity. This was indeed unclear in the previously presented model. We have now clarified this point by representing the 393-419 region of β-arrestin2, for which we do not make any hypothesis .As you requested, we now provide an in vitro kinase assay, which shows that MEK1 induces phosphorylation of purified β-arrestin2 and its canonical substrate Erk2 with comparable efficacies. This new result is illustrated on