Itch is a distinct aversive sensation that elicits a strong urge to scratch. Despite recent advances in our understanding of the peripheral basis of itch, we know very little regarding how central neural circuits modulate acute and chronic itch processing. Here we establish the causal contributions of defined periaqueductal gray (PAG) neuronal populations in itch modulation in mice. Chemogenetic manipulations demonstrate bidirectional modulation of scratching by neurons in the PAG. Fiber photometry studies show that activity of GABAergic and glutamatergic neurons in the PAG is modulated in an opposing manner during chloroquine-evoked scratching. Furthermore, activation of PAG GABAergic neurons or inhibition of glutamatergic neurons resulted in attenuation of scratching in both acute and chronic pruritis. Surprisingly, PAG GABAergic neurons, but not glutamatergic neurons, may encode the aversive component of itch. Thus, the PAG represents a neuromodulatory hub that regulates both the sensory and affective aspects of acute and chronic itch. The supraspinal mechanisms involved in itch specific sensation are not well understood. Here the authors identify a subpopulation of neurons in the periaqueductal gray that modulate chronic itch in rodents While acute itch is posited to play an important physiological role as a defense mechanism, chronic itch disorders are disabling and often difficult to treat. Chronic itch (pruritus) disorders affect 15% of the population are often associated with neuropathy, skin disease, and organ dysfunction and are the most frequent cause of visits to dermatologists2. Antihistamines are the first-line treatment for itch, but fail to control most forms of chronic pruritus2. Recently, non-histaminergic pathways for itch have been identified. Effective alternative therapies that target non-histaminergic pathways are not yet available5.Itch is a distinct unpleasant sensation that evokes a strong desire to scratch5. However, whether itch and pain information are integrated into the same or different neuronal populations is currently a matter of controversy. In agreement with the concept of shared processing nodes, recent studies support the notion of a robust itch modulatory system within regions of the nervous system that are related to nociception13. For example, scratching the skin reduces the firing rate of primate spinothalamic tract (STT) neurons responding to pruritogens, but does not alter neuronal responses to painful stimuli14. In addition, inhibition or transection of the upper cervical spinal cord reduces scratch-evoked inhibition of spinal cord neuronal activity induced by pruritic stimuli, suggesting that the inhibitory effect of scratching is due to the engagement of a strong descending supraspinal influence on spinal itch processing15. The nature of this supraspinal itch modulatory system is not known, but the similarities in anatomical pathways for pain and itch provide a framework for testable hypotheses.The neural pathways that signal itch and pain appear remarkably similar, and significant advances have recently been made to tease apart itch and pain circuitry at the level of the primary afferent fibers and the spinal cord18. The ventrolateral periaqueductal gray (vlPAG) has been shown to regulate several complex behaviors including pain19 and is a major site of endogenous opioid-induced pain suppression20. Electrical stimulation of the PAG produces profound analgesia22 by modulating spinal nociceptive transmission in part by descending projections to the rostral ventromedial medulla (RVM), which sequentially projects to the spinal cord26. It has been demonstrated in cats that27 the magnitude of the response to a pruritic stimulus is greatly diminished following decerebration, suggesting the existence of a descending itch modulation system. Consistent with this idea, electrical stimulation of the PAG results in inhibition of spinal neurons responsive to intracutaneous delivery of histamine28, and this inhibition is lost upon termination of PAG stimulation,. Although the importance of the PAG in pruritic modulation is established, very little is known regarding how the PAG participates in pruritic processing. An understanding of the midbrain neural circuits and transmitters that underlie the suppression of itch may identify novel targets for the treatment of pruritus, in a manner similar to the advancement of opioid receptor pharmacology from our understanding of the role of the PAG in pain modulation32.Neuroimaging studies suggest that the periaqueductal gray, an evolutionarily conserved structure in the midbrain, may mediate the suppression of itch induced by noxious counter-stimuli such as cold and scratchingHere, we use pharmacological, cell type-specific chemogenetic manipulations and optical imaging approaches to functionally identify and dissect the involvement of specific PAG neuronal subpopulations in the modulation of acute and chronic itch. The results that follow highlight the bidirectional modulation of scratching behaviors by PAG neurons, with opposing roles of glutamatergic and GABAergic subpopulations in mediating the sensory component of itch. Further, we identify a unique role for GABAergic PAG neurons in the encoding or modulation of itch-related aversion.+ channel blocker) into the vlPAG to block neuronal activity during pruritogen-induced scratching. Bilateral lidocaine microinjection into the vlPAG significantly attenuated scratching evoked by intradermal injection of the non-histaminergic pruritogen chloroquine (200 µg/50 µl)  Fig. . Vehicleµl) Fig. .Fig. 1Ph33 were microinjected bilaterally into the vlPAG  vectors carrying neuron-specific stimulatory (hM3Dq) or inhibitory (hM4Di) DREADDs fused with mCherry (AAV8-hSyn-hM3Dq/hM4Di-mCherry)PAG Fig. . In animPAG Fig. ,. This rPAG Fig. . As a co34.Itch is an unpleasant emotional experience in humans. We adapted a conditioned place aversion (CPA) paradigm to evaluate the unpleasant affective component of itch in mice . In contrast, animals expressing the stimulatory DREADD hM3Dq in the vlPAG showed no aversion to the chloroquine + CNO-paired chamber, . These results demonstrate that activation of hM3Dq in vlPAG neurons blocks place aversion to chloroquine and suggest that activation of vlPAG neuronal circuitry attenuates the unpleasant affective component of itch.Having demonstrated that chemogenetic activation of vlPAG neurons suppresses chloroquine-induced scratching, we next asked whether chemogenetic activation of vlPAG neurons would abolish place aversion to chloroquine. We transduced vlPAG neurons bilaterally with AAV8 vectors carrying either the stimulatory DREADD (hM3Dq) fused with mCherry or control AAV8 EGFP. After free access to both chambers on day 1, mice were conditioned for 3 days, by administering either saline or chloroquine. On conditioning days, mice were pre-treated with either saline or CNO (i.p) 1 h before being paired in the specified chambers with saline or chloroquine injection Fig. . When te37. The chemogenetic manipulations of vlPAG neuronal activity in the prior experiments do not distinguish between these two populations. To investigate the potential for differential roles of vlPAG GABAergic and glutamatergic neurons in the regulation of itch behaviors, we used fiber photometry38 to measure bulk Ca2+ dynamics in a cell type-specific manner in awake, behaving mice. By expressing the genetically encoded calcium indicator, GCaMP6s, in GABAergic (Vgat-IRES-Cre) or glutamatergic (Vglut2-IRES-Cre) neurons, we can measure neuronal activity-dependent population fluorescence changes in specific cell types within the vlPAG while mice are engaged in pruritogen-induced scratching. We virally expressed GCaMP6s in Vgat+ or Vglut2+ vlPAG neurons by injecting Cre-dependent AAV (AAV-DJ EF1a-DIO-GCaMP6s) into the vlPAG of Vgat-Cre or Vglut2-Cre mice. An optical fiber was implanted unilaterally above the vlPAG to measure changes in Ca2+ dynamics during chloroquine-evoked scratching  into the vlPAG of Vgat-IRES-Cre mice  resulted in a significant reduction of spontaneous scratching behavior produced by AEW treatment  for 7 daysent Fig. . In VgatWe next tested whether chemogenetic activation of vlPAG GABAergic neurons would block the aversive component of chloroquine-induced itch. We performed CPA to chloroquine in Vgat::hM3Dq and Vgat::mCherry mice. During conditioning, mice were pre-treated with either saline or CNO (i.p) 1 h before being placed in the saline or chloroquine paired chamber Fig. . When al+ neuronal activity observed during scratching (fiber photometry recordings above) suggest a potential causal relationship between activity of these neurons and chloroquine-evoked scratching behaviors  resulted in a significant decrease in chloroquine-evoked scratching compared to baseline Fig. . The CNOWe further analyzed whether inhibition of vlPAG glutamatergic neurons can affect scratching behaviors in the AEW dry skin model of chronic itch. Vglut2::mCh-injected and Vglut2::hM4Di-injected mice showed robust spontaneous scratching seven days after AEW treatment. Chemogenetic inhibition of vlPAG glutamatergic neurons (Vglut2::hM4Di) with CNO  resulted in a significant reduction of spontaneous scratching behavior produced by dry skin Fig. . CNO admTo determine if chemogenetic inhibition of vlPAG glutamatergic neurons can block the aversive component of chloroquine-induced itch, we performed CPA to chloroquine in Vglut2::hM4Di and Vglut2::mCherry mice Fig. . In cont42.To determine whether global manipulation of PAG neurons modulates pruritic processing via descending control at the level of the spinal cord, we performed activity-dependent mapping studies using cFos labeling . We found chloroquine injection induced cFos expression in the spinal cord, and that global chemogenetic activation of PAG neurons suppressed this chloroquine-induced induction of spinal cFos expression Fig. , whereas27. Recent studies have acknowledged the presence of defined neural circuits underlying itch processing in the brainstem, supporting this hypothesis43. It has been demonstrated that electrical stimulation of the PAG suppresses responsiveness of spinal cord neurons to histamine28, but the neural subtypes involved in modulating itch processing have never been identified. Our initial experiment to test for a role of the vlPAG in itch processing in mice utilized local pharmacologic blockade of neuronal activity (lidocaine), and supported the above studies suggesting a role for the PAG in itch modulation. This approach lacks cell-type specificity, and additionally inhibits axons coursing through or nearby the injections site. Our findings using global (not cell-type-specific) DREADD manipulation of vlPAG neurons demonstrate that activation of vlPAG neurons results in robust suppression of chloroquine-evoked scratching, while inhibition of vlPAG neurons results in profound potentiation of chloroquine-evoked scratching. This approach eliminated the possible stimulation or inhibition of axons coursing through the PAG that confounds studies utilizing either electrical stimulation-induced activation or silencing via cooling or local anesthetic administration. However, the experiments that followed demonstrate diverse roles for different cell types in the vlPAG, in a manner not easily predicted from global electrical, pharmacologic, or chemogenetic approaches described here and previously.More than 70 years ago, the brainstem was shown to play a crucial role in itch modulation46. It is unclear how distinct neuronal populations integrate and modulate different sensations. We and others have shown that the vlPAG GABAergic and glutamatergic neurons differentially modulate pain neurotransmission51. Itch and pain exhibit a reciprocal relationship at the behavioral level. For example, itch is suppressed by the mild noxious mechanical stimulus of scratching52. Given this inverse relationship of itch and pain and these identified differential roles for these neuronal populations in the vlPAG, we investigated the role of GABAergic and glutamatergic vlPAG neurons in the processing of itch. We observed that vlPAG GABAergic and glutamatergic neurons exhibited opposing effects on pruritic information processing. Activation of vlPAG GABAergic neurons resulted in decreased chloroquine-induced itch while their inhibition led to an increase in chloroquine-induced itch. Conversely, activation of vlPAG glutamatergic neurons resulted in a profound increase in chloroquine-induced itch whereas inhibition of these cells resulted in suppression of chloroquine-induced itch. Similar results were observed in a model of chronic itch, where activation of vlPAG GABAergic neurons or inhibition of vlPAG glutamatergic neurons resulted in attenuation of spontaneous scratching behavior produced by chronic dry skin. These results indicate that decreased activity in vlPAG GABAergic and/or increased activity of glutamatergic neurons may underlie central sensitization of itch in the context of chronic itch. It is likely that the vGlut2 + PAG neurons we studied here overlap with the Tac1+ neurons studied by Gao et al.42. They found that activating these neurons produced spontaneous scratching, and as such it is possible that Tac1+ neurons are small subset of vGlut2 + PAG neurons. Taken together, we show evidence for the presence of functionally divergent cell types in the vlPAG that modulate itch-evoked behavioral output and the affective component associated with pruritus. Thus, although pain and itch are processed and transmitted by similar neuroanatomical substrates at peripheral, spinal and supraspinal sites, they are discriminated and processed distinctly at the cellular level.The PAG comprises a diverse group of neuronal subpopulations that express a variety of neurotransmitters and neuropeptides54. Recently studies have shown that inhibition of vlPAG GABAergic neurons or activation of glutamatergic neurons results in antinociception, whereas activation of vlPAG GABAergic neurons or inhibition of glutamatergic neurons leads to hypersensitivity to painful stimuli36. Interestingly, these results together with our current findings demonstrate that vlPAG GABAergic and glutamatergic neurons process both pain and itch signals inversely. The activity profile of GABAergic and glutamatergic neurons was regulated inversely during chloroquine-evoked scratching, as determined here by measuring Ca2+ dynamics. The activity of these neuronal populations is consistent with the behavioral results of our chemogenetic experiments in scratching behavior, suggesting that in vivo calcium imaging faithfully recapitulates the activity of GABAergic and glutamatergic vlPAG neurons during pruritic processing. A drawback of fiber photometry is that it captures the averaged activity across a population of neurons, which might lead to missing or masking any heterogeneity in the responses of individual neurons.The vlPAG is known to have neuronal subtypes that exhibit different firing patterns in response to noxious stimuli2+ dynamics of Vgat and Vglut2 neurons are consistent with a key role of these neurons in regulating itch processing.We found that vlPAG Vgat+ and Vglut2+ neurons showed opposing responses during chloroquine-evoked scratching in our photometry recordings. Chloroquine evoked scratching inhibits GABAergic neurons and activates glutamatergic neurons. Interestingly, in GABAergic neurons basal activity is elevated just preceding chloroquine evoked scratching, while this phenomenon is not seen during spontaneous scratching bouts. What would be the underlying cause for this? We propose that GABAergic neuronal activity is encoding the sensory and motivational aspects of itch and this activity is suppressed when scratched, possibly contributing to relief of itch. An assumption in this model is that relief of itch by scratching is responsible for suppression of activity of GABAergic neurons. Interestingly, we observed rapid elevated activity in the Vglut2+ neurons subsequent to scratching. These differences in the modulatory effects of GABAergic and glutamatergic neurons on itch response suggests the presence of distinct microcircuit features. It is possible that inhibition of Vgat+ neurons might disinhibit the Vglut2 +ve neurons subsequent to scratching during chloroquine-evoked itch. The Ca46. Even among the subpopulations studied here, there are certain to be further subclassifications based on anatomy  or neurochemistry . It is unlikely that all the GABAergic and glutamatergic vlPAG neurons show the same response to pruritic processing. Prior work50 suggests that GABAergic neurons in the PAG are mostly interneurons, and we did not find any downstream projections from these neurons in our studies. PAG glutamatergic neurons are output neurons of the PAG. Differences in the modulatory effects of GABAergic and glutamatergic neurons on itch responses suggest the presence of distinct microcircuit features. Though not addressed directly in our study, it is possible that activation of Vgat neurons that encode itch might directly inhibit the Vglut2 neurons to suppress chloroquine evoked ongoing itch. Future studies may discover additional heterogeneity in GABAergic and glutamatergic neuronal subtypes based on their gene expression and their anatomical downstream targets.The opposing and unique actions of glutamatergic and GABAergic neurons in the modulation of itch identified here emphasize the importance of using cell-type-specific approaches. This also suggests that further dissection of these neuronal subtypes should be undertaken. We have chosen to assess potential unique roles for glutamatergic and GABAergic neurons for the reasons mentioned above, however the PAG comprises multiple neuronal populations expressing several neurotransmitters and neuropeptidesThere were some notable discrepancies between our behavioral data obtained from pharmacological and chemogenetic manipulation of global PAG activity compared to cell-type specific PAG manipulations  or to the amygdala neurons that encode aversion36 , Slc17a6tm2Lowl (Vglut2-ires-Cre), and C57BL/6J mice were purchased from Jackson Laboratories and colonies were established in our facilities. Litters and animals were randomized at the time of assigning experimental conditions for the whole study. Experimenters were blind to treatment and genotype.All experiments were conducted in accordance with the National Institute of Health guidelines and with approval from the Animal Care and Use Committee of Washington University School of Medicine. Mice were housed on a 12-h light-dark cycle (6:00 a.m. to 6:00 p.m.) and were allowed free access to food and water. All animals were bred onto C57BL/6J background and no more than 5 animals were housed per cage. Male littermates between 8–12 weeks old were used for experiments. Slc32a112 particles/ml, Lot number: AV5359 and Lot date: 6/13/2013), hM4Di-mCherry ,and hSyn-eGFP  was used as used as control for non Cre dependent studies in C57 mice. Cre-dependent hM3Dq-mCherry and hM4D-mCherry  Control mCherry  was used to express in the Vgat and Vglut2 Cre mice. All vectors were packaged by the University of North Carolina Vector Core Facility. All vectors were aliquoted and stored in −80 °C until use. We functionally verified and validated DREADD constructs we used in this study in our prior publication36.Purified and concentrated adeno-associated viruses coding for Cre-independent hM3Dq-mCherry  where surgical anesthesia was maintained using 2% isoflurane. Mice were kept on a heating pad for the duration of the procedure. Preoperative care included application of sterile eye ointment for lubrication, administration of 1 mL of subcutaneous saline and surgery site sterilization with iodine solution. A small midline dorsal incision was performed to expose the skull and viral injections were performed using the following coordinates: vlPAG, −4.8 to 4.9 mm from bregma, +/− 0.3 to 0.4 mm lateral from midline and 2.7 to 2.9 mm ventral to skull. Viruses were delivered bilaterally, using a stereotaxic mounted syringe pump (Micro4 Microsyringe Pump Controller from World Precision Instruments) and a custom 2.0 µL Hamilton syringe. Injections of 150 nL of the desired viral vectors into the area of interest were performed at a rate of 1 µL per 10 min. We allowed for a 10-min period post injection for bolus diffusion before removing the injection needle. Postoperative care included closure of the cranial incision with sutures and using veterinary tissue adhesive and application of topical triple antibiotic near the incision site. Animals were monitored while on a heating pad until they full recovery from the anesthetic.The surgical protocol was the same as the described above for viral injections. Stainless steel guide cannulas (8 mm) were implanted at the vlPAG and fixed to the skull using two bone screws  and dental cement. An 8 mm stylet was used to avoid guide cannula clogging. Mice were allowed to recover for 7 days. Microinjections of 0.25 μl 4% lidocaine  diluted in 0.9% saline were performed 15 min before pruritic agent induced scratching behavior was assessed. Mice were later euthanized, and cannula placements were verified using cresyl violet nissl stain. Animals in which cannula’s were not placed in the vlPAG are excluded from the study.36. Three weeks later mice were injected with  clozapine N-oxide  2 h before doing behavioral experiments and data were collected between the second-hour and third-hour post-injection. All baselines for pruritic responses were recorded 2 weeks after the virus injections and one week prior to the CNO administration. We performed dose response studies of CNO to determine the optimal dose for use in behavioral experiments. Based on these dose-finding studies, we selected 5 mg/kg CNO as a dose of CNO that produces near maximal effects in Cre dependent DREADD-expressing animals and showed no signs of behavioral affects in control vector-expressing animals. For non-Cre dependent studies, we observed that a dose above 1 mg/kg CNO was lethal, so we used 1 mg/kg CNO dose for both activation and inhibition of vlPAG neurons.For non-specific chemogenetic control of vlPAG neurons, C57BL/6J mice were injected with non-Cre dependent control eGFP, hM3Dq or hM4Di viruses. For subpopulation specific chemogenetic control of vlPAG neurons Vgat-ires-Cre and Vglut2-ires-Cre mice were injected with Cre dependent control mCh, hm3Di or hm4Dq viruses. DREADD constructs used in this study were validated previously for their functional expression in the PAG, including their ability to increase (hM3Dq) or decrease (hM4Di) neuronal firing in slices from animals expressing these viral constructs41. For chemogenetic manipulations, CNO was administered before placing the mice in the plexiglass behavioral boxes and chloroquine  induced scratching behavior was performed 90 min after the CNO administration.The nape of the neck mice was shaved one day prior to experiments. Mice then were placed in clear plexiglass behavioral boxes for at least two hours for acclimation for baselinesTo model dry skin-mediated itch, spontaneous scratching behavior was induced by treatment twice daily with an acetone/diethylether (1:1) solution for 15 s followed by water for 30 s on the nape of the neck. Following seven days of AEW treatment, spontaneous behavior was video recorded for two hour pre-CNO and post-CNO. Scratch bouts directed to the nape of the neck and rostral back were counted in 5-min intervals during blinded off-line analysis.61. Scores were calculated by subtracting the time spent in the chloroquine-paired compartment, post-test minus the pre-test. To test the effect of DREADD hM3Dq activation on chloroquine-induced place aversion, mice injected with AAV8 hM3Dq–mCherry and AAV8 EGFP were allowed free access to all three chambers for 30 min on the pre-conditioning day (day 1). On day 2, 3, and 4, both mice cohorts received a saline injection (50 µl) on the nape of the neck and on the mouse caudal back, and this chamber is paired with systemic saline injection 2 h before they were placed in the compartment in the morning and a chloroquine injection (400 µg/50 µl) on the nape of the neck and on the mouse caudal back and this chamber is paired with systemic CNO injection 2 h before they were placed in the compartment in the afternoon. To test the effect of DREADD hM3Dq activation on chloroquine-induced place aversion, the mice were allowed free access to the three compartments on day 5 for 30 min. Scores were calculated by subtracting the time spent in the chloroquine-paired compartment, post-test minus the pre-test.CPA was performed using an unbiased, counterbalanced three-compartment conditioning apparatus. Each chamber had a unique combination of visual, properties . On the pre-conditioning day (day 1), mice were allowed free access to all three chambers for 20 min. Behavioral activity in each compartment was monitored and recorded with a video camera and analyzed using Ethovision 8.5 (Noldus) or ANY-Maze softward. Mice were randomly assigned to saline and chloroquine compartments and received a saline injection (50 µl) on the nape of the neck and on the mouse caudal back, in the morning and a chloroquine injection (400 µg/50 µl) on the nape of the neck and on the mouse caudal back in the afternoon, at least 4 h after the morning training on 3 consecutive days . To enhance the association of chloroquine induced scratching behavior with the paired chamber, we administered chloroquine and left the mice in their holding cage for 4 min, then placed them in the paired chamber during the time of the peak scratching response (20 min in the chamber). To assess for place aversion, the mice were then allowed free access to all three compartments on day 5 for 30 min13 particles/ml, Stanford vector core). Fiber optic probes were unilaterally implanted above the left vlPAG . After 4 weeks of viral expression, mice were handled and acclimated, tethered as during imaging, for 7 days in the test behavioral chamber. On test day, mice were habituated with the tethered fiber optic patch cord  in the test chamber (15 × 15 cm) for 30 min and injected with chloroquine (200ug/50ul) on the nape of the neck and recordings were performed.For in vivo calcium imaging of vlPAG GABAergic and glutamatergic neurons, we injected vlPAG with Cre-dependent GCaMP6s  was bandpass filtered (FMC1 + (405/10) -(475/28)_(525/45)_FC), Doric lenses) and delivered to the vlPAG to excite GCaMP6s. The emitted light was bandpass filtered (FMC1 + _(405/10)-(475/28)_(525/45)_FC), Doric lenses) and sent to a photoreceiver to detect the signal . The signal from the photoreceiver was recorded using a RZ5P real-time processor (TDT). Data were acquired at 10 kHz and then demodulated at 211 and 537 Hz. The demodulated signal was then low-pass filtered (4 Hz) in a custom MATLAB script. The extracted 405 nm signal was then scaled to fit the GCaMP signal for the recording session. To isolate the movement-corrected GCaMP signal from channel, we subtracted the signal at 405 nm from the 475 nm GCaMP signal. dF/F was obtained by dividing the final signal with its mean value. Behavioral event timestamps associated with chloroquine evoked scratching behavior were scored and aligned with GCAMP signal in the MATLAB script to create pre-stimulus and peri-stimulus time bins. To obtain pre-stimulus and peri-stimulus chloroquine evoked scratching events, if the scratching events happened to close to each other in a 30 s window, they we combined and called as one event. Z-score was obtained by subtracting the mean of the GCaMP signal from the bin value of the GCaMP signal and dividing it with the standard deviation of the bin value of the GCaMP signal.A fiber optic patch cord was used to connect to the fiber implant and deliver light to excite and record the GCAMP signal using a custom-built fiber photometry rig. The fiber photometry recording setup was built with some modifications to previously described specifications62. Slides containing spinal cord sections were fixed in 4% paraformaldehyde, dehydrated, and pretreated with protease IV solution for 30 min. Sections were then incubated with target probes for mouse cFos . Following probe hybridization, sections underwent a series of probe signal amplification steps (AMP1–4) followed by incubation of fluorescent probes , designed to target the specified channel associated with the probes. Slides were counterstained with DAPI and coverslips were mounted with Vectashield Hard Set mounting medium (Vector Laboratories). Images were obtained on a Leica TCS SPE confocal microscope (Leica), and Application Suite Advanced Fluorescence (LAS AF) software was used for analyses. To quantify number of cFos+ve cells, we counted DAPI-stained nuclei that coexpress minimum of five cFos puncta as an cFos+ve cell. We did not include any cFos puncta that does not overlay on top of the DAPI-stained nuclei as part of our analysis.For non-specific chemogenetic control of vlPAG neurons, C57BL/6 J mice were injected with non-Cre dependent control mCh, hM3Dq or hM4Di viruses. Three weeks later mice were injected with CNO (BML-NS105 from Enzo life sciences) 2 hrs before chloroquine was injected on the nape of the neck. Thirty minutes post chloroquine administration, mice were rapidly decapitated, cervical spinal cords were dissected and flash frozen in −50 °C 2-methylbutane and stored at −80 °C for further processing. Coronal sections of the spinal cord corresponding to the cervical level, were cut at 15 μM at −20°C and thaw-mounted onto Super Frost Plus slides (Fisher). Slides were stored at −80 °C until further processing. FISH was performed according to the RNAScope® 2.0 Fluorescent Multiple Kit v2 User Manual for Fresh Frozen Tissue 66.Adult mice were deeply anesthetized using a ketamine/xylazine cocktail and then perfused with 20 ml of with phosphate-buffered saline (PBS) and 4% paraformaldehyde (weight/volume) in PBS . Brains were carefully removed, post fixed in 4% PFA overnight and later immersed in 30% sucrose for at least 48 h. Tissues were mounted in OCT while allowing solidification of the mounting medium at −80 °C. Using a cryostat, 30μm tissue sections were collected and stored in PBS1× 0.4% sodium azide at 4 °C. After washing the sections in PBS1×, we blocked using 5% normal goat serum and 0.2% Triton-X PBS 1× for one hour at room temperature. Primary antibodies against mCherry  and GFP  were diluted in blocking solution and incubated overnight at 4 °C. After three 10-min washes in PBS1×, tissues were incubated for one hour at room temperature with secondary antibodies (Life Technologies: Alexa Fluor488 donkey anti rabbit IgG (1/300); Alexa Fluor 488 goat anti rabbit (1/300); Alexa Fluor 555 goat anti mouse (1/300)) and Neurotrace  at room temperature. Three PBS1× washes followed before sections were mounted with Vectashield (H-1400) hard mounting media and imaged after slides cured. Images were obtained on a Nikon Eclipse 80i epifluorescence microscopet test was used for comparing paired observations, we evaluated for normality using the D’Agostino and Pearson omnibus normality test for all datasets. Therefore, only when normality could be assumed, we used a parametric test to analyze out data. If normality could not be assumed, a nonparametric test or a Wilcoxon matched pairs text was used to evaluate differences between the means of our experimental groups. Two-way ANOVA was used for comparing between different control and treatment groups. Bonferroni’s post hoc tests were used (when significant main effects were found) to compare effects of variables. A value of p < 0.05 was considered statistically significant for all statistical comparisons.Throughout the study, researchers were blinded to all experimental conditions. Exclusion criteria for our study consisted on a failure to localize expression in our experimental models or off-site administration of virus or drug. At least 3 replicates measurements were performed and averaged in all behavioral assays. The number of animals used is indicated by the “N” in each experiment. When paired Supplementary InformationPeer Review FileSource Data


Homologous recombination deficiency (HRD) measured using a genomic signature for loss of heterozygosity (LOH) predicts benefit from rucaparib in ovarian cancer. We hypothesized that some oesophagogastric cancers will have high-LOH which would be prognostic in patients treated with platinum chemotherapy.Diagnostic biopsy DNA from patients treated in the REAL3 trial was sequenced using the Foundation Medicine T5 next-generation sequencing (NGS) assay. An algorithm quantified the percentage of interrogable genome with LOH. Multidimensional optimization was performed to identify a cut-off dichotomizing the population into LOH-high and low groups associated with differential survival outcomes.Of 158 available samples, 117 were successfully sequenced; LOH was derived for 74 of these. A cut-off of 21% genomic LOH defined an LOH-high subgroup  who had median overall survival (OS) of 18.3 months (m) versus 11m for the LOH-low group . Progression free survival (PFS) for LOH-high and LOH-low groups was 10.7m and 7.3m . Sensitivity analysis censoring operated patients (n=4), demonstrated OS of 18.3m vs. 10.2m , p=0.02; PFS was 10.5m vs. 7.2m , p=0.09 for LOH-high and LOH-low.HRD assessment using an algorithmically derived LOH signature on a standard NGS panel identifies oesophagogastric cancer patients with high LOH who have prolonged survival when treated with platinum chemotherapy. Validation work will determine the signature's predictive value in patients treated with a PARP inhibitor and with platinum chemotherapy. BRCA mutant tumours using a synthetically lethal approach with poly ADP ribose polymerase (PARP) inhibitors has resulted in beneficial effects for patients in ovarian, breast and prostate cancer [BRCA wild-type HRD tumours have high levels of “genomic scarring”, which arises from the use of error prone DNA repair pathways when homologous recombination is compromised. One method for quantifying the amount of genomic scarring is to assess the extent of loss of heterozygosity  across the tumour genome. Determination of HRD using LOH may have clinical implications; in the ARIEL2 Part 1 (NCT01891344) trial of previously treated ovarian cancer, BRCA wild-type patients with high levels of LOH (LOH-high) treated with the PARP inhibitor rucaparib were more likely to respond to rucaparib therapy and had longer progression free survival compared to rucaparib-treated patients who were not LOH-high [Homologous recombination (HR) is a complex process requiring the coordinated function of a number of genes products in order to repair double-stranded breaks in DNA . This pre cancer –7. Howeve cancer . Such BRLOH-high .Oesophagogastric cancer is a platinum sensitive disease in which several genomic and proteomic biomarkers associated with DNA repair defects have been identified. These include ATM loss, The Cancer Genome Atlas (TCGA) chromosomally unstable (CIN) subtype and a putative BRCA mutational signature –16. TherDetails of the REAL3 trial have been previously described . In brieOut of a total of 553 REAL3 patients 158 formalin-fixed paraffin embedded (FFPE) tissue blocks with high tumour content (>30%) were available; these were sent for NGS analysis to Foundation Medicine (FM). There was no significant difference in clinicopathological characteristics, progression free survival or overall survival between patients who underwent sequencing for LOH assessment and those without see . Of the There was no difference in the proportion of samples which were successfully sequenced from each tumour site; however junctional and oesophageal tumours were more likely to have LOH successfully inferred than stomach cancer (73% and 66% vs 50% respectively). Table The median percentage of genomic LOH inferred for all tumours was 11.9% (n=74)  were classified as LOH-high. At this cut off, the median overall survival (OS) was 18.3 months for the LOH-high subgroup compared to 11 months for the LOH-low subgroup. Using a Cox proportional hazards model, we derived the OS hazard ratio to be 0.55 (95% CI 0.19-0.97), p= 0.10. At the same LOH cut off, progression free survival (PFS) was 10.7 months for the LOH-high group compared to 7.3 months for the LOH-low group. Using a Cox proportional hazards model, the PFS HR was 0.61 (95% CI 0.21 – 1.09), p=0.09. Figures MANOVA of these clinicopathological variables potentially associated with progression free and overall survival showed that of these parameters only disease extent have a statistically significant impact on both progression free and overall survival Table . ExtendeBRCA2 (n=1), ATM (n=6), ATR (n=1), CHK2 (n=1). Additionally, 101 of 117 (86%) samples had mutations in the TP53 gene. There was no significant difference in %LOH, OS, and PFS between the TP53 mutated and wildtype populations, other mutant populations were considered too small to analyse separately.HRD can also be caused by variants in genes of the HR pathway that render the protein function impaired. Categories of variants that are deleterious to protein function include protein truncating mutations, splice site mutations, homozygous deletions and large protein truncating rearrangements. The Foundation Medicine T5 NGS assay has been validated to detect these classes of variants. In the REAL3 samples sequenced here, several deleterious mutations in genes of the HR pathway were detected and are detailed in Table In this analysis we identify a group of oesophagogastric cancer patients treated in the REAL3 trial who have high levels of genomic LOH measured using an algorithmic approach to DNA sequencing data performed on tissue obtained from routine diagnostic biopsies. Using a cut-off of 21% genomic LOH to define LOH-high, we demonstrate that patients with higher levels of LOH have a trend towards longer overall survival than those who are not LOH-high. When a small number of patients who underwent potentially curative surgery were censored in a sensitivity analysis, these findings reached statistical significance , p=0.02) These findings are of interest for two reasons. Firstly, although the predictive power of the biomarker cannot be determined in the absence of a control group, high LOH may identify patients who are more likely to benefit from platinum based chemotherapy. Secondly, as LOH-high patients with ovarian cancer benefit from PARP inhibition more than LOH–low patients, it is possible that high LOH could be a biomarker predictive of sensitivity to PARP inhibitors in oesophagogastric cancer patients. While these hypotheses require prospective validation, they could lead to more effective biomarker selected therapies for oesophagogastric cancer, a disease which currently has a dismal prognosis , 19.Platinum based chemotherapy is a standard first line treatment for patients with advanced oesophagogastric cancer –23. To dThe genomic and transcriptomic landscape of oesophagogastric cancer has been intensively explored recently. The recently published oesophageal TCGA data suggests that oesophageal adenocarcinoma is molecularly almost indistinguishable from chromosomally unstable gastric cancer . These cRecent seminal work has demonstrated that chromosomal instability drives metastasis independently of aneuploidy and has described increased levels chromosomal instability in a cohort of matched primary tumour and brain metastases, in addition to a series of matched breast cancer primaries and metastases . TherefoPARP inhibitors have a well-defined role in ovarian cancer, the value of PARP inhibition in oesophagogastric cancer is less clear. The Phase III randomized phase III GOLD trial failed to demonstrate a statistically significant overall survival advantage for olaparib treated patients in intention to treat trial population , although this may be in part due to a statistical correction for multiple primary endpoints . NotablyOne limitation of our study is the small proportion of tumours from the total trial population which were successfully sequenced; prior biomarker studies on the same population had exhausted much of the available tissue . In partIn conclusion, in this study we present our results from deriving a genomic signature for high LOH in oesophagogastric cancer patients treated with platinum based chemotherapy in the REAL3 trial, which we found to be prognostic for survival in this patient population. The results of the current study can be considered preliminary, and analysis of a larger cohort is necessary in order to provide further validation on the hypothesized clinical significance of LOH. Specifically, we acknowledge that the small size of our data set made it impossible to separate the data into a training data set and one to test the LOH cutoff prospectively. We plan to do this in the phase II randomized PLATFORM trial (NCT02678182) which is currently running in the United Kingdom and which will have recruited almost one thousand patients when fully accrued. As immuno-oncology therapy moves to the fore, tumours with high levels of genomic scar which elicit robust immune responses may also be candidates for immune checkpoint therapy, and it is possible that combining PARP inhibition and checkpoint inhibitor therapy could provide long term benefits for selected patients , 37.From the REAL3 cohort (n=553) pre-treatment tumour biopsies (tissue blocks) with high tumour content (>30%) were selected by a pathologist. All patients included in this analysis had given informed consent for translational research. The REAL3 trial was conducted under national and local ethical approvals.The Foundation Medicine T5 next-generation sequencing assay  was used to calculate the percentage of genomic LOH in pretreatment biopsies, a minimum DNA input of 200ng is recommended for the assay . This asThe primary endpoint of the study was to determine a cut-off for LOH which separated patients into two groups (LOH-high and LOH-low) which were associated with distinct survival outcomes. To set a cutoff for separating samples high in genomic LOH from those low in genomic LOH, we performed a multi-dimensional optimization of parameters across all possible LOH cutoffs across the range of observed LOH values . Across Multivariate analysis of variance (MANOVA) was used to identify variables that could potentially confound any findings related to survival. The investigated potentially confounding variables were: age, gender, tumour site, disease extent, WHO performance status, histological subtype, and treatment group. To control for the effects of potentially confounding variables we performed an extended confounding analysis and applied groupwise statistical tests to the parameters above in the two groups, LOH-high and LOH-low. In order to homogenise the patient population we performed a sensitivity analysis in which patients who underwent potentially curative surgery after chemotherapy were censored for progression free and overall survival at the time of surgery.


It is well known that contracting the upper limbs can affect spinal reflexes of the lower limb muscle, via intraneuronal networks within the central nervous system. However, it remains unknown whether neuromuscular electrical stimulation (NMES), which can generate muscle contractions without central commands from the cortex, can also play a role in such inter-limb facilitation. Therefore, the objective of this study was to compare the effects of unilateral upper limb contractions using NMES and voluntary unilateral upper limb contractions on the inter-limb spinal reflex facilitation in the lower limb muscles. Spinal reflex excitability was assessed using transcutaneous spinal cord stimulation (tSCS) to elicit responses bilaterally in multiple lower limb muscles, including ankle and thigh muscles. Five interventions were applied on the right wrist flexors for 70 s: (1) sensory-level NMES; (2) motor-level NMES; (3) voluntary contraction; (4) voluntary contraction and sensory-level NMES; (5) voluntary contraction and motor-level NMES. Results showed that spinal reflex excitability of ankle muscles was facilitated bilaterally during voluntary contraction of the upper limb unilaterally and that voluntary contraction with motor-level NMES had similar effects as just contracting voluntarily. Meanwhile, motor-level NMES facilitated contralateral thigh muscles, and sensory-level NMES had no effect. Overall, our results suggest that inter-limb facilitation effect of spinal reflex excitability in lower limb muscles depends, to a larger extent, on the presence of the central commands from the cortex during voluntary contractions. However, peripheral input generated by muscle contractions using NMES might have effects on the spinal reflex excitability of inter-limb muscles via spinal intraneuronal networks. More than a century has passed since Ernst Jendrássik found that clenching the teeth or the fists facilitates tendon taps  in the lower limbs  can be used to generate muscle contraction by depolarizing axons of alpha motor neurons beneath the electrodes placed on the skin surface over the muscles or nerves, which provides a rich afferent input to central nervous system without voluntary effort  to investigate remote effect facilitation of lower limb muscles during bilateral upper limb contraction tasks  sensory-level NMES; (2) motor-level NMES; and (3) voluntary contraction. Moreover, the second objective of this study was to investigate how combining NMES and voluntary contraction of the upper limb may affect facilitation of spinal reflex excitability: (4) voluntary contraction and sensory-level NMES; (5) voluntary contraction and motor-level NMES. Afferent feedback during voluntary contractions plays an important role in modulating the spinal reflex excitability . All participants had no history of neuromuscular and sensory disorders. All participants gave written informed consent in accordance with the Declaration of Helsinki. The experimental procedures were approved by the local institutional ethics committee at The University of Tokyo.Mmax) of right flexor carpi radialis (FCR)  were evoked: (i) pre; (ii) during; and (iii) post each intervention . Biphasic rectangular stimulation waves were applied with a 400 µs pulse width and 20 Hz frequency via circular surface electrodes (2 cm diameter) positioned on the brachium (cathode) and on the motor point (anode), which was searched by the experimenter using a pen electrode to locate a motor point that evoked wrist flexion effectively. Pulse width and frequency used in this study were determined based on previous studies, which suggested that these parameters can effectively stimulate the sensory nerves , but remain fatigue resistant  . Responses recorded from tibialis anterior (TA), soleus (Sol), vastus medialis (VM), and biceps femoris (BF) muscles bilaterally  using surface electromyography (EMG) electrodes . A long reference electrode was placed around the circumference of the knee . The anode electrode (5 × 5 cm) was positioned on the midline of the abdomen and the cathode electrode (7.5 × 10 cm) was placed on the lumbar spine. The optimal site of the cathode electrode was determined based on the location that induced larger responses simultaneously in all recorded lower limb muscles  of the right (stimulated) FCR muscle was representatively recorded before and after each experimental condition by stimulating the median nerve using surface electrodes placed between the biceps brachii and brachialis. The anode was placed proximally and the cathode distally, with 1.5 cm separation. Five Mmax responses were elicited before and after each experimental condition, with 5 s interval between the stimuli  and band-pass filtered at 15–3000 Hz  without any additional processing  and to test differences of background EMG  between pre, during, and post each intervention in each muscle. When Friedman test resulted in a significant effect, Wilcoxon signed-rank tests with Bonferroni correction were used to compare the mean values. Moreover, Wilcoxon signed-rank test was also used to compare the first and second responses of the paired-pulse stimulation and the Mmax amplitudes before and after each intervention. Significant level was set at p < 0.05 for all tests.Normality of data was first tested using the Shapiro–Wilk test and the tests indicated that not all data were normality distributed. Thus, Friedman test, a non-parametric equivalent for repeated-measure analysis of variance (ANOVA), was used to test differences of spinal reflex amplitudes before, during, and after each intervention in each muscle to find the change of Mmax) of the FCR muscle throughout the experiment are shown in Fig. Mmax during the whole experiment . Moreover, Mmax amplitude before and after each intervention was also not significantly different. Overall, the result suggested that wrist flexor muscles were not affected by peripheral fatigue through the experiment . Results comparing spinal reflexes during different experimental interventions are summarized in Fig. For all muscles, background EMG activity was not significantly different between pre, during, and post for each intervention , elicited by transcranial magnetic stimulation (TMS) over the motor cortex to evaluate the corticospinal excitability, are facilitated by remote muscle activations  facilitated the spinal reflex excitability of lower limbs, as we hypothesized, but the facilitation effect appeared only in contralateral thigh muscles  Fig. . HoweverChange of spinal reflex excitability of ankle dorsiflexors  was observed bilaterally even during voluntary muscle contraction assisted with motor-level NMES , but in the plantar flexors , spinal reflex excitability was not modulated Fig. . These eIn rehabilitation after spinal cord injury, significantly larger improvements of walking function and the cervicolumbar connectivity were found in the training group that included combined arm and leg cycling compared to the group that trained using only leg cycling (Zhou et al. Mmax amplitude. It is unfeasible to consistently evoked M-wave using tSCS in multiple muscles simultaneously due to a large stimulus current requirement. However, the stimulation intensity of tSCS was adjusted to be on the ascending part of the recruitment curve in all recorded muscles simultaneously. Moreover, baseline amplitude was measured before each intervention (Fig. Some limitations should be considered in the present study. First, Courtine and colleagues reported that spinal reflex responses of the TA muscle evoked by tSCS could be contaminated by Sol muscle activity (Courtine et al. ion Fig. b and it ion Fig.  (see Dation Fig. . Third, ion Fig. . Howeverion Fig. . TherefoWe used NMES to the median nerve unilaterally to investigate the effect of peripheral afferent information on the spinal reflex excitability of lower limbs, compared to voluntary unilateral muscle contractions of the upper limb. Our results showed that voluntary flexion of the wrist facilitated the spinal reflex excitability of distal ankle muscle bilaterally. Meanwhile, the spinal reflex excitability of contralateral thigh muscles was facilitated by wrist flexion induced by motor-level NMES. Overall, the present results suggest that the central motor commands are important for the facilitation of spinal reflex excitability of remote limb muscles by voluntary muscle contractions and imply that distal ankle muscles have a stronger connectivity with the forearm compared to the thigh muscles in the interlimb facilitation. The afferent information evoked by NMES on upper limb has different effects compared to voluntary muscle contractions on the spinal reflex excitability of lower limbs. Nonetheless, involvement of upper limb muscles either by voluntary contractions or NMES can likely help in rehabilitation of walking.


Stereotactic Ablative Radiotherapy (SABR) is an effective treatment that improves local control for many tumours. However, the role of SABR in gynecological cancers (GYN) has not been well-established. We hypothesize that there exists considerable variation in GYN-SABR practice and technique. The goal of this study is to describe clinical and technical factors in utilization of GYN-SABR among 11 experienced radiation oncologists.A 63 question survey on GYN-SABR was sent to 11 radiation oncologists (5 countries) who have published original research, conducted trials or have an established program at their institutions. Responses were combined and analyzed at a central institution.Most respondents indicated that salvage therapy (non-irradiated or re-irradiated field) for nodal (81%) and primary recurrent disease (91%) could be considered standard options for SABR in the setting of inability to administer brachytherapy. All other indications should be considered on clinical trials. Most would not offer SABR as a boost in primary treatment off-trial without absolute contraindications to brachytherapy. Multi-modality imaging is often (91%) used for planning including PET, CT contrast and MRI. There is a wide variation for OAR tolerances however small bowel is considered the dose-limiting structure for most experts (91%). Fractionation schedules range from 3 to 6 fractions for nodal/primary definitive and boost SABR.Although SABR has become increasingly standard in other oncology disease sites, there remains a wide variation in both clinical and technical factors when treating GYN cancers. Nodal and recurrent disease is considered a potential indication for SABR whereas other indications should be offered on clinical trials. This study summarizes SABR practices among GYN radiation oncologists while further studies are needed to establish consensus guidelines for GYN-SABR treatment. Radiation plays an important role in the treatment of gynecological cancers. In both the definitive and adjuvant setting, local radiotherapy to pelvic primaries and lymph nodes have been shown to improve local control and outcome in different gynecological malignancies such as cervical, uterine and vulvovaginal tumours –6. BrachSABR is well-known to be an effective radiation treatment technique for multiple tumour types and for various indications. Malignancies of the the lung, liver, brain, and the spine have well-established protocols and consensus in regard to treatment indications, technique and dose , 9. SABRA systematic review of SABR treatment for GYN cancers showed that this technique, although not standardly indicated, has been used for multiple GYN indications in a small number of single arm prospective clinical trials and several smaller retrospective series –12. ThesA 63 question survey was sent to 11 radiation oncologists who practice and have published on GYN SABR in 5 different countries. Participating institutions included Department of Radiation Oncology, Sunnybrook Health Science Centre and Odette Cancer Centre, , National Cancer Institute, , Delaware County Memorial Hospital/Philadelphia Cyberknife , twenty-first Century Oncology , University Hospital Zuerich, , Department of Radiation Oncology, Northeastern Radiation Oncology, , Soonchunhyang University Seoul Hospital , Institut Oncologic Teknon, , Case Western Reserve University School of Medicine, , Universita degli Studi di Messina, , University of Texas Southwestern Medical Center, . The survey included 63 questions in 5 parts. Topics of the questionnaire were pertaining to clinical and technological factors of GYN SABR technique. These included questions on the indications for GYN SABR, the eligibility criteria, pre-treatment investigation, treatment planning protocol, target delineation, dose targets and constraints and treatment response evaluation. Surveys were sent via e-mail and responses were collected and evaluated at a central institution (OCC). The questionnaire was completed during the period of January 2017 to March 2017. Responses were presented as percentages of agreement for each question among the 11 participants.All participants completed the survey. The median number of GYN SABR patients treated in total per participant was 40 (5–125). SABR for GYN malignancies is used most commonly for recurrent disease  in patients who have had prior radiation and/or have contraindications to brachytherapy or surgery (64–91%). Salvage pelvic and para-aortic nodal SABR was highly cited in this international survey as a common practice in patients with locally advanced gynecological cancers without prior radiation (82%).SABR is not typically used as an alternative to brachytherapy but could be considered in the setting of clinical trials (64%). Primary SABR boost can be used when there is a contraindication to brachytherapy (73%). The indications of GYN SABR from the survey are summarized in Table Applying a size constraint for GYN SABR was common in setting of cervical/vaginal boost and nodal SABR (64%). Tumour size constraints varied between 5 and 8 cm or 90–600 cc. Nodal size limits varied from 3 to 5 cm or 125–600 cc. An age restriction is not common for SABR treatment in GYN cancers (91%) however some respondents felt that patients should generally have a performance status of 0–2 ECOG or KPS (64%).It is recommended that pathological confirmation is obtained prior to starting SABR treatment (82%), however, histological subtype (eg. squamous cell carcinoma vs. adenocarcinoma) does not typically influence SABR treatment decision. (100%).Contraindications to GYN SABR include brachytherapy eligibility (64%), tumour in close proximity to small bowel (46%), and previous high dose radiation/brachytherapy to the target site (46%).Multi-modality imaging for staging and tumour assessment is typically performed prior to SABR treatment, including computerized tomography (CT) (82%), positron emission tomography (PET-CT) (82%), magnetic resonance imaging (MRI) (91%). For planning CT, slice thickness of 2 mm or less is used (82%). Most common organ motion strategies include fiducial markers (82%) and bladder protocol (82%). Intravenous contrast is often used with CT simulations (73%) with no routine use of premedication. Common treatment delivery techniques include VMAT (73%) and cyberknife (64%), with some centres using both techniques. Photon energy range between 6 MV to 18 MV.Immobilization devices used include leg immobilizers (36%) and full body fix vaclock (64%). Implanted fiducials and kV x-rays are common for image guidance (64%). Other techniques include cone-beam CT (45%), robotic tracking (45%) and daily orthogonal KV x-rays (36%), with some centres using multiple techniques. Daily pre-treatment imaging and online correction is used. Rotation correction errors of > 1–3 mm are recommended (91%), with repeat imaging after couch adjustment (73%). Interfraction monitoring with fiducials is also recommended but post-treatment imaging is rare (45%). Checking pre-treatment imaging is important before each fraction (82%).When chemotherapy is indicated, most respondents treat sequentially (73%), with a break of at least 1–3 weeks. The remaining respondents do not typically treat with SABR around the the time of systemic therapy.When considering GYN SABR for recurrent nodal disease, the clinical target volume (CTV) is considered to be the gross nodal disease with an expansion (73%) whereas 9% considers the CTV to be the gross tumour volume (GTV) only and another 9% considers the CTV to be the GTV plus the nodal region at risk. A planning target volume (PTV) expansion of 2–7 mm is typically applied. For the CTV of recurrent primary disease (eg. cervix), most treatment consider this to be the GTV or an expansion on the GTV (36–46%). Of the 8 participants that have used SABR as a boost treatment, 3 defined the target as the GTV + cervix + grey zones while the rest only treated the GTV +/− expansion.PTV is often trimmed from organs at risk (OARs) to avoid overlap (82%), however a safety margin on OARs, or planning-at-risk-volumes (PRVs), is not typically used. Coverage of PTV is most commonly 95%. For CTV, 95–100% and GTV coverage is commonly 100%. Most common parameter used from planning optimization is conformity indices (82%).Target dose prescription is prescribed to the periphery of the target (73%). Maximum hotspots can range between 105 and 200%. The isodose line covering the periphery can range from 65 to 90%.(α/β = 10) (26.8–71.2). For recurrent primary SABR, median 40.4 EQD2 (α/β = 10) (27–71.2). For boost SABR after standard pelvic radiation, Median 36.75 EQD2 (α/β = 10) (15.6–60). Fractionation schedules for different centres participating in this study are summarized in Table Different fractionation doses and schedules are in practice for definitive SABR for recurrent disease and boost SABR treatment after external beam radiation. Fractionation regimens typically are between 3 and 5 fractions. For recurrent nodal SABR, median dose is 36 Gy EQD2 OARs most commonly limited to a dose constraint in GYN SABR include rectum, bladder, sigmoid, bowel, spinal cord and kidney with most recommending small bowel as the dose limiting organ (91%). A summary of dose constraints used for GYN SABR are presented in Table SABR follow-up done in clinic every 3, 6 or 12 months with gynecological exam and investigations, including MRI, PET and CT contrast. Imaging is typically done 2–4 months after treatment to limit false positives from radiation changes. Local control is assessed through RECIST criteria or negative PET scan. Post-treatment biopsy is rare.To our knowledge, this is the first study summarizing the practice and technological considerations of GYN SABR among experts that practice and have published on this technique , 10–21. As GYN SABR is becoming increasingly common in practice, aligning international protocols may help guide the establishment of this technique as one of the standard options for certain clinical indications. In the systematic review by Mendez et al., six major clinical indications of GYN SABR were found in the published literature . These iIt should be noted that the survey results don’t reflect the more standard North American approach of salvage extended field nodal radiation and more trials are needed in this area before such practice is changed. With the emergence of SABR treatment for oligometastatic disease, there is increasing interest in this strategy for GYN tumours . SeveralSome would consider boost treatment with SABR, however, most agree that this is only an option when there is a contraindication to brachytherapy. Although SABR can deliver high conformal doses of radiation to the target, its ability to deliver high central pelvic doses is still inferior to brachytherapy with current technologies and this can lead to suboptimal outcomes , 30. In Imaging and treatment protocols were found to vary amongst the participating centres. GYN radiation oncologists have generally been late to adopt conformal techniques due to concerns of significant degrees of organ motion such as the cervix which has been shown to move as much as 4 cm in the anterior-posterior directions . TherefoCTV definitions were generally similar among participants with some using brachytherapy contouring guidelines to define targets in the central pelvis. Dose fractionation varied for different indications and varied among participants with most using 3–5 fractions for SABR treatment. There was also a wide variation of OAR constraints used with most treatment target constraints being similar in both the de novo and re-irradiation setting. These constraints reflect the institutional practices of the respondents and are not intended to serve as a consensus guideline for GYN SABR planning. Well-researched dose constraints that are not specific to GYN SABR include the AAPM-101 report and the more recent updated UK Consensus , 34. FurGYN SABR is commonly used in specialized centres around the world. Although SABR can deliver high-dose conformal radiation to targets, most experts agree that it is only used as a definitive boost treatment when brachytherapy is contraindicated. SABR for nodal and pelvic tumour salvage is becoming one of the common treatments and dose-fractionation and OAR tolerances vary among different centres. Future collaborative studies and clinic trials are warranted to better define the role and to standardize the practice of SABR for gynecological malignancies.


Surprisingly, steroid-structure-specific preferences do not require cell viability, implying a passive mechanism, and occur across cells derived from multiple tissue types. Physiologic relevance is suggested by structure-specific preferences in human prostate tissue compared with serum. On the other hand, the presence of serum proteins in vitro blocks much, but not all, of the passive accumulation, while still permitting a substantial amount of active accumulation for certain steroids. Our findings suggest that both passive and active uptake mechanisms make important contributions to the cellular steroid uptake process. The role of passive, lipophilicity-driven accumulation has previously been largely unappreciated, and its existence provides important context to studies on steroid transport and action both in vitro and in vivo.Steroid hormones and their respective nuclear receptors are essential mediators in numerous physiologic and pathophysiologic processes, ranging from regulation of metabolism, immune function, and reproductive processes to the development of hormone-dependent cancers such as those of the breast and prostate. Because steroids must enter cells before activating nuclear receptors, understanding the mechanisms by which cellular uptake occurs is critical, yet a clear understanding of these mechanisms has been elusive. It is generally assumed that diffusion-driven uptake is similar across various steroids whereas an elevated cellular concentration is thought to reflect active uptake, but these assumptions have not been directly tested. Here we show that intact cells rapidly accumulate free steroids to markedly elevated concentrations. This effect varies widely depending on steroid structure; more lipophilic steroids reach more elevated concentrations. Strong preferences exist for 3β-OH, Δ After one-hour incubation at 37°C, media were removed and transferred to 2 mL microcentrifuge tubes . Cells were washed once with D-PBS, then scraped into 800 μL D-PBS and transferred to 2 mL microcentrifuge tubes. Cell samples were subjected to 3x freeze-thaw cycles to lyse cells. We observed that some fraction of the steroids in samples adhered to plastic tubes, so 700 μL 1:1 ethyl acetate:isooctane (1:1) was added to each media and cell sample, which prevented this adhesion. Total contents of each microcentrifuge tube were then transferred to scintillation vials containing Liquiscint scintillation fluid . Total radioactive signal in each sample was assayed by LS 6000IC scintillation counter . In each experiment, cells from two untreated wells were scraped into D-PBS and volumes of cell pellets were measured using PCV packed cell volume tubes . The volumes of the pellets from the two wells were averaged and the average volume was used for cellular concentration (counts/μL) calculations from that experiment. Typical volumes of pellets were approximately 3 to 5 μL. All experiments were performed with biological duplicates and repeated independently at least twice. In these experiments specifically for JEG-3 cells treated with pregnenolone, [3H]-labeled pregnenolone was supplemented with 10 μM unlabeled pregnenolone because we observed that rapid conversion by enzyme 3βHSD of pregnenolone to progesterone in JEG-3 cells . Note that the effect on pregnenolone uptake of adding a saturating concentration of pregnenolone was, however, tested in LNCaP cells in a separate experiment  were plated in 6-well plates coated with poly-L-ornithine. The day after seeding, medium was replaced with 800 μL serum-free medium and cells were treated with [ells see  section ment see  section.3H]-labeled steroids. After media and cell samples were collected and cells were lysed via 3x freeze-thaw cycles, 10 μL internal standard  was added to each sample and samples were transferred to glass test tubes for liquid-liquid extraction. 2 mL methyl tert-butyl ether (MTBE) was added to each tube; tubes were vortexed for 5 minutes and centrifuged  and the MTBE layer was collected and then dried under nitrogen gas and reconstituted in 120 μL 50% methanol/water (v/v). The extracted steroids were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS). Briefly, the extracted steroids were injected onto a Shimadzu Nexera 2 UHPLC system , and the steroids were separated on a C18 column  using a gradient starting from 20% solvent B [acetonitrile/methanol  containing 0.2% formic acid] for 4 min and then to 75% solvent B for 10 min, followed by 95% solvent B for 3 min. The steroids were quantified on a Qtrap 5500 mass spectrometer  using ESI in positive ion mode and multiple reaction monitoring (MRM) using characteristic parent → daughter ion transitions for the specific molecular species monitored. Androstene-3,17-dione-2,3,4-13C3  was used as an internal standard for calibration of steroids in each sample. Data acquisition and processing were performed using MultiQuant . The peak area ratio of the analyte over the internal standard was used for quantification. Each sample run included a calibration curve with standards for data quantification using the analyte/internal standard peak area ratio.Experiments assaying uptake by mass spectrometry were performed in the same way as normal steroid uptake experiments except that 100 nM unlabeled steroids  were used in lieu of [3H]-labeled steroids  and incubated for one hour at 37°C on tube rotators. In an additional set of experiments, cells were treated with 2 nM [3H]-labeled steroids with or without the addition of 1 μM unlabeled steroids. Cells were pelleted by 5 min. 250 g centrifugation in an Allegra 6R centrifuge (Beckman-Coulter). Under this protocol, dead HMC-1-8 cells, unlike live HMC-1-8 cells and live or dead LNCaP and JEG-3 cells, did not form good pellets, so HMC-1-8 cells were not included in this experiment. After pelleting, media were removed from tubes and transferred to 2.0 mL microcentrifuge tubes. Cell pellets were collected in 800 μL D-PBS and transferred to 2.0 mL microcentrifuge tubes. Cell samples were subjected to 3x freeze-thaw cycles to lyse cells. 800 μL 1:1 ethyl acetate:isooctane (1:1) was added to each media and cell sample to prevent steroid adhesion to tubes. Total contents of each microcentrifuge tube were then transferred to scintillation vials containing scintillation fluid and total radioactive signal in each sample was assayed by scintillation counter. In each experiment, volumes of cell pellets from untreated tubes (two tubes each of live and dead cells) were measured using PCV tubes.For parallel live and dead cell experiments, cells  were aliquoted into 1.5 mL microcentrifuge tubes  in 1 mL serum-free media. For the dead cell condition, tubes were heated to 90–95°C for 15 min. Cells were confirmed to be dead by trypan blue assay using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific). Live and dead cells were then treated in parallel with [3H]-labeled steroid with or without the addition of 1 μM unlabeled steroid. Additionally, because we observed that serum did not wash out of dishes as readily as culture media, parallel experiments were performed with no cells and a correction was applied to the results with cells by subtracting the steroid content of the residual serum in the control experiments from the steroid content of the cell samples.Experiments testing uptake in the presence of serum were performed in the same way as normal steroid uptake experiments except that culture media were replaced with human serum  or charcoal-stripped fetal bovine serum . Steroid concentrations for these experiments were 2 nM [Each experimental treatment resulted in corresponding measurements of total cellular signal and total media signal. The cellular signal was divided by the cell volume from PCV tube measurement to obtain the cellular concentration (counts/μL). The cellular signal and media signal were added together and divided by the media volume to obtain the original media concentration upon treatment (counts/μL). The cellular concentration was divided by the original media concentration to obtain the ratio reported in the graphs throughout the results section.5 cells per well) were plated in poly-L-ornithine coated 12-well plates with 10% charcoal stripped fetal bovine serum (CSS) phenol red-free RPMI and incubated for 48 h at 37°C. Cells were then incubated with 10 nM [3H]-steroid in transport buffer  at 37°C for the indicated time points. At each time point, the media from the wells was collected, and the cells were washed with ice cold transport buffer. Cells were lysed with 1% TritonX-100 in PBS, collected, and protein concentration determined by BCA assay. Samples were added to 3 mL Liquiscint, and radioactivity was measured on a liquid scintillation counter. Results were graphed as /protein concentration in units of μL/mg using GraphPad Prism 5 .The uptake of DHEA, pregnenolone, progesterone, and Δ4-androstenedione (AD) was measured in LNCaP cells as previously described with minor modifications . Briefly13C-AD) were extracted from the tissue samples after homogenization with methanol followed by solid phase extraction. Serum samples (100 μL) were subject to liquid liquid extraction; the steroids and the internal standards were extracted with MTBE evaporated to dryness under N2 then reconstituted with 200 μL of 50% methanol. Steroids and their corresponding internal standards were ionized using electrospray ionization in positive mode on an AB Sciex 5500 Qtrap coupled with Shimadzu Nexera UPLC station.All patient samples were obtained using an Institutional Review Board-approved protocol. Matching serum and tissue samples from patients with localized prostate cancer undergoing radical prostatectomy were subject to liquid chromatography tandem mass spectrometry analysis (LC-MS/MS). Steroids  and their corresponding internal standards  were added to 50 mL buffer). Cells were incubated on ice for 15 minutes, then lysed by using a 1 mL syringe to pass through a 27 gauge needle 10 times, then left on ice for an additional 20 minutes. Lysate was then centrifuged for 5 min at 720 g and 4°C on a 5424R centrifuge  and supernatant was collected and transferred to a fresh tube. This supernatant was centrifuged for 5 min at 10,000 g and 4°C, the new supernatant was transferred to a 14 x 25 mm Quick Seal centrifuge tube (Beckman Coulter), and additional fractionation buffer was added to fill the tube . Samples were then centrifuged for 1 h at 100,000 g and 4°C on an Ultima L-100 XP ultracentrifuge with SW 41Ti rotor (Beckman Coulter). The supernatant was removed, forming the cytosol fraction, and the pellet, forming the membrane fraction, was collected in 550 μL fractionation buffer. Samples were analyzed by mass spectrometry as described above.Subcellular fractionation was performed using a modified version of a previously described protocol , 24. LNCFig 1A) and the effects of steroid structural differences on uptake , human cell lines derived from prostate, breast, and placenta were treated in vitro with nine different [3H]-labeled steroids in parallel: pregnenolone, progesterone, DHEA, Δ4-androstenedione (AD), testosterone (T), dihydrotestosterone (DHT), DHEA sulfate (DHEA-S), cortisol, and cortisone . Uptake of estradiol and estrone was tested in a second set of experiments, in parallel with pregnenolone and DHEA to aid in comparison to the initial set of experiments . Although there were minor differences between cell lines, the overall uptake trends were surprisingly consistent across the three cell lines. All tested steroids had substantially greater concentrations in the cells after 1 hour than in the culture media. Our major observation was that pregnenolone reached the highest concentrations of any steroid, with concentrations up to roughly 100 times the original treatment concentrations in the culture media, and that cells have strong preferences for 3β-OH, Δ5-steroids  vs. 3-keto, Δ4-structural features  and for progestogens vs. androgens.To test cells’ tendency to take up steroids  in cells that were heat killed prior to steroid treatment. In these experiments, cells in suspension in microcentrifuge tubes were killed by heating to 90–95°C, removed from the heat, and then treated with steroids in parallel to live cells that underwent the same procedure except for the heating. Cell death of the heat-killed cells was verified by trypan blue assay . Because heating cells to 90–95°C would result in both permeabilization of membranes -labeled steroids . These results support the conclusion that a passive mechanism drives cellular preferences for progestogen and 3β-OH, Δ5-steroid uptake.Our finding that cells consistently have a large capacity for steroid uptake that differs greatly in magnitude for different steroids raises the question of the nature of the uptake mechanism. To test whether the uptake involves active cellular processes, we assayed uptake of six -labeled steroid via active mechanisms  would be stopped either by heat-killing the cells prior to steroid incubation or by incubating the cells with an excess concentration of unlabeled steroid to saturate active transport and/or binding. We again treated cells with six steroids  under four simultaneous conditions: 2 nM [3H]-labeled steroid with no unlabeled steroid in live cells, 2 nM [3H]-labeled steroid with 1 μM unlabeled steroid in live cells, and the same two sets of concentrations in dead cells. The results ( were suggestive of contributions of both active and passive accumulation in the prostate cancer (LNCaP) cells in which this experiment was performed. When active accumulation was not blocked , pregnenolone, progesterone, and testosterone exhibited higher levels of steroid uptake than when active accumulation was blocked ; DHT also showed a trend toward higher uptake. By contrast, AD and DHEA exhibited no evidence of active accumulation. These findings are consistent with a report that active uptake by prostate cancer cells of testosterone is competed with by progesterone and DHT but not by DHEA -labeled steroid both without and with 1 μM unlabeled steroid and performed the experiments with cells in charcoal-stripped fetal bovine serum so that endogenous steroids present in non-charcoal-stripped serum would not interfere with the uptake of [3H]-labeled steroid. The results , as in our previous experiment with cells in serum-free media, showed that pregnenolone, progesterone, testosterone, and DHT appear to have both active and passive components of cellular uptake whereas DHEA and AD appear to only have passive components. Comparing Fig 10B to Fig 3, it can be seen that both passive and active accumulation were diminished by the presence of serum proteins, but the relative contribution of active mechanisms appears to be larger when serum proteins are present. For example, in Fig 3 (no serum proteins present), about one-third of the 2 nM [3H]-labeled pregnenolone that accumulated in cells did so by active mechanisms; by contrast, in Fig 10B (serum proteins present), about 80% of the 2 nM [3H]-labeled pregnenolone that accumulated in cells did so by active mechanisms.To directly test the capacity for cells to accumulate free steroids, all the in vitro experiments described thus far were performed using serum-free media, but in vivo a large proportion of steroid molecules are bound to serum proteins . To test5-steroids over steroids with 3-keto, Δ4-structural features and progestogens over androgens. One caveat of the findings is that the initial sets of experiments  used [3H]-labeled steroid treatment amounts determined by activity, not by molarity, and therefore molar concentrations of different steroids were not exactly the same, but subsequent experiments utilized standardized molar concentrations of steroids and resulted in similar trends. The cellular accumulation of steroids occurs rapidly, within minutes, with a longer time course for steroids that reach higher concentrations. Rapid metabolism of pregnenolone by placental choriocarcinoma cells suggests that the cellular accumulation of steroids is not merely occurring at the cell surface or plasma membrane but in the interior of the cell. The structure-specific preferences that we observed in vitro persist in concomitantly obtained prostate tissue and peripheral blood patient samples, suggesting our findings may be relevant to human physiology. Our in vivo analysis surveyed a more limited set of steroids than our in vitro studies and only included one tissue type (prostate), a limitation that could be addressed by further studies. The degree of cellular steroid accumulation is predicted by the octanol-water partition coefficient of a steroid; more lipophilic steroids are taken up more by cells. More lipophilic steroids accumulate more in the membrane fraction of fractionated cells, and modifying a steroid to alter its lipophilicity correspondingly alters its cellular uptake, further supporting the hypothesis that cellular uptake of free steroids is largely driven by lipophilicity-based accumulation in lipophilic compartments of cells. Lastly, we showed that serum proteins prevent most of the passive uptake of steroids by cells, suggesting that passive cellular uptake of steroids may only draw from the free, not the protein-bound, steroid pool, while active uptake mechanisms may have some ability to draw from the protein-bound steroid pool.In this study, we have demonstrated that cells from multiple tissue types uniformly have a tendency to accumulate free (non-protein-bound) steroid molecules at elevated concentrations. Although active mechanisms appear to contribute to the uptake of certain steroids, blocking active mechanisms by heat-killing cells prior to steroid treatment or by saturation reveals strikingly large passive components of steroid uptake. All steroids we tested reach elevated cellular concentrations, but the degree to which this occurs is dependent on steroid molecular structure; for example, cells preferentially take up 3β-OH, Δow-measured lipophilicity has been known, in certain fields, for many years [The tendency of lipophilic molecules to accumulate in lipids  in a manner dependent on log Kny years . In thatny years , in vivony years , or intany years , 36 leveny years , 38 and The apparent discrepancy between the elevated prostate tissue to peripheral blood concentration ratios we observed in vivo, an environment in which circulating steroids are mostly protein-bound, and the much less elevated ratios we observed when in vitro experiments were performed in serum is an issue that requires further study. Others have reported comparisons of serum and tissue concentrations for certain steroids and tissues with varying results. For example, two different groups , 40 repolower than the total steroid concentrations in blood. Given that cells do have a strong tendency to passively accumulate free steroids, as our results show, the binding of steroids to serum proteins may serve to ensure that a sufficient proportion of steroid molecules can circulate through the body rather than being caught in a sink of lipophilic compartments in cells and tissue, similar to a hypothesis put forth by Mendel et al. [Interestingly, although heightened tissue concentrations have been cited as evidence for active uptake, it is rarely pointed out that, because most steroids circulate with >95% of molecules bound to serum proteins , if celll et al.  regardinFig 6) may be of particular relevance to the effects of this and possibly other mutations [Because steroid hormones must enter cells before carrying out most of their diverse actions, cellular uptake of steroids is relevant to many areas of physiology and disease, among them hormone-driven cancers such as those of the breast and prostate. Prostate cancer (PCa) growth is driven by testosterone and the more potent DHT activation of the AR. The mutant AR ligand binding domain (T877A) present in LNCaP cells and found in castration-resistant prostate cancer (CRPC) patients is strongly activated by additional ligands other than testosterone and DHT , includiutations . Additioutations , furtherFig 10). On the other hand, the rapid time course of diffusion, especially for less lipophilic steroids , could make it difficult to accurately measure the steroid content of cells, as a too thorough washing of cells could almost completely eliminate the passively generated cellular concentration of a less lipophilic steroid.Our findings have important implications for the design and interpretation of experiments in which cells are exposed to steroids in vitro. In such studies, cells are typically either in serum-free media or media with 10% serum. If a physiologic concentration of serum proteins were present, steroid uptake into cells, particularly passive uptake, would be greatly diminished  imply the presence of a large pool of intracellular pregnenolone that is available as a substrate for enzyme 3βHSD1. This suggests the possibility that steroid molecules are continuously moving into and out of lipophilic compartments to re-equilibrate their concentrations as a reaction progresses.A study by Chisari et al. of GABAreceptor . The stureceptor . IntriguFig 4), Chisari et al. showed that more lipophilic neuroactive steroids have longer time courses of modulatory effects and of cellular accumulation. Additionally, a study on transport of steroids across a Caco-2 cell monolayer showed that the transport occurred via passive diffusion and that there was a negative correlation between lipophilicity and the rate of diffusion across the monolayer [Also consistent with our findings, in particular those on half-maximal uptake times  provide additional evidence for active mechanisms, but suggest that these active mechanisms, which may be specific to certain steroids and types of cells and may include carrier-mediated transport processes as well as binding of steroids to nuclear receptors, occur in addition to the high capacity for passive uptake of free steroids that cells possess. Whereas elevated cell or tissue concentrations of steroids have been interpreted as evidence for active uptake [Fig 1E vs. 1C and 1D) are suggestive that the differences may point to differential active uptake on top of a more uniform passive uptake background. In a study in Drosophila it was suggested that a membrane transporter is required for cellular steroid uptake and steroid hormones in Drosophila cannot enter cells via passive diffusion [Others have described evidence for active mechanisms mediating cellular steroid uptake , 55–57. e uptake , 34, ouriffusion . It is iS1 Fig(A) and dead (B) LNCaP cells incubated in tubes and treated with 100 nM unlabeled steroid. All graphs show mean ± SD from one representative experiment with biological duplicates and all experiments were performed at least twice. For all graphs, the uptake of pregnenolone was significantly greater than all other steroids .Ratios of cellular concentrations to original treatment concentrations in culture media for three different steroids in live (TIF)Click here for additional data file.S2 FigFig 6.Box and whisker plot of sex hormone binding globulin (SHBG) concentrations in the peripheral blood patient samples used in (TIF)Click here for additional data file.


Callosobruchus maculatus that is known to exhibit SA genetic variance in fitness. We measured sex-specific competitive lifetime reproductive success  in >500 sex-by-genotype F1 combinations and found that segregating genetic variation in fitness exhibited pronounced contributions from dominance variance and sex-specific dominance variance. A closer inspection of the nature of dominance variance revealed that the fixed allelic variation captured within each strain tended to be dominant in one sex but recessive in the other, revealing genome-wide SSDR for SA polymorphisms underlying fitness. Our findings suggest that SA balancing selection could play an underappreciated role in maintaining fitness variance in natural populations.The maintenance of genetic variance in fitness represents one of the most longstanding enigmas in evolutionary biology. Sexually antagonistic (SA) selection may contribute substantially to maintaining genetic variance in fitness by maintaining alternative alleles with opposite fitness effects in the two sexes. This is especially likely if such SA loci exhibit sex-specific dominance reversal (SSDR)—wherein the allele that benefits a given sex is also dominant in that sex—which would generate balancing selection and maintain stable SA polymorphisms for fitness. However, direct empirical tests of SSDR for fitness are currently lacking. Here, we performed a full diallel cross among isogenic strains derived from a natural population of the seed beetle  Evolution requires genetic variation, but selection will tend to fix whichever alleles confer the highest fitness, depleting the genetic variation upon which it acts. Sexually antagonistic (SA) genetic variation—in which alternative alleles have opposite fitness effects in the sexes—can generate balancing selection that maintains genetic variation for fitness if the alleles that benefit a given sex are also dominant in that sex. Here, we show that the SA genetic variation underlying fitness in a well-known seed beetle population exhibits these beneficial reversals of dominance, suggesting SA selection may commonly maintain heritable genetic variation for fitness throughout the genome. One of the most longstanding challenges for evolutionary biologists has been to explain the maintenance of genetic variance in fitness –7. SelecSexually antagonistic (SA) selection can cause alternative alleles to have opposite fitness effects in males and females –18 and hThe capacity for SA selection to generate balancing selection that results in stable polymorphisms for fitness drastically increases with sex-specific dominance reversal (SSDR) –42, in wThe ability of SSDR to promote the maintenance of SA polymorphisms for fitness has been known for decades , yet theThe full diallel cross  is the pC. maculatus that is known to exhibit pronounced SA genetic variance in fitness  and strain 3 [as sire]) and was measured as the total number of F2 offspring emerging from these assays—i.e., the competitive lifetime reproductive success of F1 individuals.Male (top right) and female (bottom right) fitness was assayed in the F(TIF)Click here for additional data file.S3 FigBayesDiallel VarPs Click here for additional data file.S4 FigaS), with strain 1 being the most female beneficial/male detrimental and strain 16 being the most male beneficial/female detrimental. Pronounced SC additive effects (a) would be represented by strains having relatively easily identifiable vertical columns (the strain’s additive contribution as a sire) and horizontal rows (the strain’s additive contribution as a dam) with a consistent shade that does not vary (much) between males and females or with contributions from other strains . Pronounced SA additive effects (aS) would be represented by easily identifiable patterns of a in one sex of a given strain with a shade toward the opposite extreme in the opposite sex of that strain . Alternatively, aS can be visualized by looking at the whole population: a subtle light-to-dark and dark-to-light gradient from top left to bottom right among the outcrossed families is apparent in males (A) and females (B), respectively, since the strains are arranged in reverse rank order of their HPD means for aS. Disruptions to the “smoothness” of this gradient—generating a more mosaic pattern—represent the basis of variance in the different forms of dominance and epistasis: b, v, and w. Although parental effects (c) were not found to have an important contribution to fitness variance in this population, they would, in principle, appear as differences between sire- and dam-specific patterns of a. SA versions of any effect would, in principle, appear as its SC counterpart pattern exhibiting the opposite shade gradient between panels A and B. HPD, highest posterior density; SA, sexually antagonistic; SC, sexually concordant.Difference between male (A) and female (B) posterior predictive means for relative fitness  in order to more easily identify patterns of inheritance among the outcrossed families see . Strains(TIF)Click here for additional data file.S5 Figβ) render this figure mostly useful for visualizing the fixed effects of S, β, and βS. The similar average shade of heterozygotes between panels A and B represents no difference in mean fitness between males and females (S). The relative shade difference between inbred parental selfs  and outbred heterozygotes represents the effect of inbreeding (β), and the difference in β between panels represents the sex-specific effect of inbreeding (βS), which was stronger in males. The inbreeding effects make it difficult to identify patterns of inheritance among the outcrossed families—this is more easily seen using relative fitness  and female (B) posterior predictive means for log fitness Click here for additional data file.S6 FigThe example shown here would be for a given sex of strain 1 (whose family means are indexed as dam×sire), in which case the covariance between the elements of the two vectors  and —corresponding to the family means for which the mother is from strain 1—would be averaged with the covariance between the elements of the two vectors  and —corresponding to the family means for which the father is from strain 1—to give a single covariance for a given sex of strain 1. This was done for each strain, for male Click here for additional data file.S7 Fig10.5061/dryad.m06s2.Log-transformed and variance-standardized mean male and female fitness for the isofemale lines from Berger and colleagues . Filled (TIF)Click here for additional data file.S1 TableBayesDiallel VarPs see  for fitn(PDF)Click here for additional data file.S2 TableBayesDiallel VarPs see  for sepa(PDF)Click here for additional data file.S3 TableP values for the overall (fixed) effects and variances (σ2) with s.e. for strain- and cross-specific (random) effects (variance components). REML, restricted maximum likelihood; s.e., standard error.Results of separate male and female REML models displaying F statistics with (PDF)Click here for additional data file.S1 TextPresentation of the results of the Bayesian analysis (and its comparison with the REML results) and further methodological information on the study population, statistical rationale, Bayesian statistical modeling, and the terminology and meaning of variance components. REML, restricted maximum likelihood.(DOCX)Click here for additional data file.S1 DataFive sheets of data described as follows. Raw data: Full diallel data depicting the “fitness” for individuals of each “sex”  in relation to the strain IDs of their fathers and mothers  and the replicate “block” to which each observation belongs. (XLSX)Click here for additional data file.