Chinchilla laniger that enabled direct perilymphatic infusion. Through this strategy we unmasked gentamicin’s potential effects on afferent calyces. The pathophysiology of the vestibular neuroepithelia after post-administration durations of 0.5 through 6 months was assessed using single-neuron electrophysiology, immunohistochemistry, and confocal microscopy. Hair cell densities within cristae central zones  and utricle peri- and extrastriola (6-months) regions were determined, and damage to calretinin-immunoreactive calyces was quantified. Gentamicin-induced hair cell loss exhibited a profile that reflected elimination of a most-sensitive group by 0.5-months post-administration (18.2%), followed by loss of a second group (20.6%) over the subsequent 5.5 months. The total hair cell loss with this gentamicin dose (approximately 38.8%) was less than the estimated fraction of type I hair cells in the chinchilla’s crista central zone (approximately 60%), indicating that viable type I hair cells remained. Extensive lesions to afferent calyces were observed at 0.5-months, though stimulus-evoked modulation was intact at this post-administration time. Widespread compromise to calyx morphology and severe attenuation of stimulus-evoked afferent discharge modulation was found at 1 month post-administration, a condition that persisted in preparations examined through the 6-month post-administration interval. Spontaneous discharge was robust at all post-administration intervals. All calretinin-positive calyces had retracted at 2 and 6 months post-administration. We found no evidence of morphologic or physiologic recovery. These results indicate that gentamicin-induced partial lesions to vestibular epithelia include hair cell loss (ostensibly reflecting an apoptotic effect) that is far less extensive than the compromise to stimulus-evoked afferent discharge modulation and retraction of afferent calyces (reflecting non-apoptotic effects). Additionally, calyx retraction cannot be completely accounted for by loss of type I hair cells, supporting the possibility for direct action of gentamicin on the afferent dendrite.Although the effects of aminoglycoside antibiotics on hair cells have been investigated for decades, their influences on the dendrites of primary afferent neurons have not been widely studied. This is undoubtedly due to the difficulty in disassociating pathology to dendritic processes from that resulting from loss of the presynaptic hair cell. This was overcome in the present investigation through development of a preparation using  The vestibulotoxic effects of aminoglycosides became apparent shortly after implementation of streptomycin therapy in the treatment of tuberculosis . Early iIn most studies of aminoglycoside-induced vestibular pathophysiology, the lesions have been extensive with damage to hair cells, supporting cells, and afferent neurons. However, particular components of the mammalian vestibular neuroepithelia are believed to exhibit greater sensitivity to the deleterious effects of aminoglycosides than others. There is evidence suggesting that type I hair cells, particularly those in cristae central zones and utriclar striolae, are the most sensitive and are the first to be compromised or eliminated after aminoglycoside administration . Lyford-Although gentamicin toxicity to vestibular hair cells has been extensively investigated, few studies have focused upon the effects on Scarpa’s ganglion neurons and their dendrites within the sensory epithelia. non-apoptotic effects. If the latter alternative is true, identifying epithelial constituents that are generally labile to other ototoxic agents, then there is hope for rehabilitation of vestibular hypofunction resulting from toxicity secondary to systemic aminoglycoside or other therapies. These issues were addressed in the present study through the development of a novel preparation enabling the use of refined gentamicin dosing that resulted in less extensive yet highly repeatable lesions than achieved in previous studies. The goal of these preparations was to use lower gentamicin doses to produce partial lesions enabling the distinction of hair cell and afferent pathology. Pathophysiologic correlates of these lesions were determined through single-afferent electrophysiology and immunohistochemical methodologies.Because the investigations by Guide for the Care and Use of Laboratory Animals , and the principles presented in the Guidelines for the Use of Animals in Neuroscience Research by the Society for Neuroscience (available from the Society for Neuroscience). All procedures were approved by UCLA’s institutional animal care and use committee.Adult male chinchillas  were used for this study. These animals were acquired, cared for, and handled in accordance with the guidelines published in the NIH n = 5/13) exhibited a slight head tilt toward the lesioned side within 2–3 postoperative days, whereas no vehicle control specimens (n = 3) exhibited head tilt. The head tilt resolved within 2 weeks after drug administration. Spontaneous nystagmus was not observed in any animal subjects.For the surgical implantation of a perilymph access port enabling direct gentamicin infusion, animals were anesthetized and placed on a platform equipped with a servo-controlled heater for core temperature maintenance (approximately 36.5°C) throughout the surgical preparation and gentamicin administration. Two anesthesia protocols were utilized during this study. For the early preparations, the protocol included administration of an intramuscular cocktail of ketamine and xylazine , followed by maintenance doses that amounted to 25% of the initial dose administered only as needed. For later preparations, isoflurane anesthesia (2–2.5%) was used exclusively. Once a surgical plane of anesthesia was achieved, the head was placed within a custom holder. A midline scalp incision was made to expose the surface of the tympanic bulla, and the bulla’s bony cap was removed to expose the middle ear. The chinchilla exhibits cavernous tympanic bullae with plenty of space between the prominent bony superior semicircular canal and the dorsal cap of the bulla. At the canal’s dorsal-most aspect, a small fenestra was carefully made into the perilymphatic space surrounding the membranous superior canal, into which a 5 mm length of 27-gauge stainless steel tubing was fit and secured with cyanoacrylate cement. The fenestra was made to provide patent access to the perilymphatic space surrounding the semicircular canal, but was not so large to allow the tubing to completely enter the superior semicircular canal and potentially occlude the duct. Once the cyanoacrylate cement cured, an epoxy-like bonding agent  was poured around the cannula to secure it in place and fix the entire preparation to the surrounding temporal bone, leaving the top 1 mm of cannula exposed. By the time the bonding agent cured (approximately 5 min), perilymph was generally visualized at the top of the cannula. The cannula was fit with polyethylene tubing (PE-20) leading to a precision syringe placed in an infusion pump. A fixed volume of treatment solution  was administered directly to the perilymphatic space over a 1-h period. Administration of 0.4 μg/μl gentamicin in 2.5 μl HBSS amounted to a total delivery of 1 μg gentamicin. The PE delivery tubing was then removed and the cannula sealed prior to replacing the bulla’s bony cap. The scalp incision was then sutured, whereupon the animal was removed from anesthesia and closely monitored until it regained sternal posture (<30 min). Upon full recovery from anesthesia, a minority of gentamicin treated specimens (2) was placed. Core body temperature was maintained at 38°C via rectal probe and a custom servo-controlled heating system. Each animal’s head was held in a stereotyped position  by a custom holder fixed to a servo-controlled rotation table. This position was maintained for each experimental preparation. Heart and respiratory rates, as well as blood oxygen saturation, were recorded at regular intervals throughout the recording session.The function of the vestibular epithelia in untreated, gentamicin-treated, or vehicle-control  preparations was assessed by electrophysiologic recording from individual afferent neurons following post-administration durations of 0.5, 1, 2, and 6 months. At the specified duration, animals were deeply anesthetized with a single intraperitoneal administration of sodium pentobarbital (50 mg/kg), which was sufficient to allow for cannulation of a jugular vein through which maintenance doses were administered as needed . A tracheotomy was performed for placement of an endotracheal tube into which a loosely fitting cannula delivering supplemental oxygen . Utricular afferents were identified as those that were unresponsive to turntable rotations, or responded at twice the stimulus frequency .Recordings from individual vestibular afferent neurons were made by exposing the right superior vestibular nerve in the region of Scarpa’s ganglion through a fenestra made in the internal vestibular meatus, approximately 1mm medial to the superior and horizontal semicircular canal ampullae. Vestibular afferent discharge was recorded using glass microelectrodes pulled to impedances of approximately 40 MΩ when filled with 1 M KCl. With the animal restrained in the custom head holder, afferent discharge was recorded in the absence of head movement stimuli  and during a broad repertoire of stimulus rotations. Afferents projecting from the horizontal and superior cristae and from the utricle could be recorded from this site in the superior vestibular nerve, which were easily distinguished in normal animals based upon their discharge modulation during manual turntable rotations. That is, in untreated preparations horizontal canal afferents were identified by increased discharge rate during turntable rotations producing utriculopetal relative endolymph flow, while superior canal afferents exhibited decreased discharge rate with similar rotations  were calculated during a 20 s recording epoch, from which the mean and standard deviation were determined. The coefficient of variation (CV) for each afferent was then computed as the ratio of ISI standard deviation to ISI mean. The perstimulus discharge spike trains were first analyzed using discrete Fourier analysis. However, upon finding that the responses in gentamicin-treated specimens were severely attenuated or non-existent, it became clear that methods were needed to objectively verify a statistical correlation between stimulus and response. To achieve greater measure of confidence in evaluating very weak responses, we adapted the frequency-domain measures for determining stimulus–response coherence computed directly from the spike trains .As in all experiments employing glass microelectrodes and axonal recordings, an inherent sampling bias exists toward the larger axons in the chinchilla’s superior vestibular nerve. Despite this potential bias, our recordings from untreated specimens suggest that it is offset with methods  resulting in a proclivity for afferents with low values of CV, indicative of smaller caliber afferents . The inhWe utilized fluorescence immunohistochemistry and confocal microscopy to visualize the cytoarchitecture of chinchilla vestibular sensory epithelia. Vestibular endorgans were prepared as whole mounts and incubated with an antibody to calretinin (anti-CALB2) to immunolabel calyx-only afferents, and, in some specimens, an antibody to β3-tubulin (anti-TUBB3) to label all calyces and nerve fibers. In contrast to murine vestibular organs in which most hair cells are calretinin-immunopositive (CALB2+) ,b, only After specified post-administration durations and electrophysiologic recording sessions, animals were euthanized  and temporal bones were quickly dissected. Temporal bones were immediately infused with 4% paraformaldehyde (in 0.1 M phosphate buffer) through the oval window, after which the roof of the vestibule (floor of the subarcuate fossa) was removed prior to incubating in fixative overnight at 4°C. After three washes in 0.1 M phosphate buffered saline , vestibular epithelia and nerve branches were microdissected. The tissues were incubated in blocking solution  for 2 h at room temperature, and then incubated 48–72 h at 4°C in a primary antibody cocktail that included combinations of the following antibodies diluted in blocking solution: 1:250 mouse anti-CALB2  or 1:250 rabbit anti-CALB2 , 1:250 rabbit anti-Class III β-TUBB3 , 1:250 rabbit anti-Myosin VI . After washing in PBS, specimens were incubated for 2 h at room temperature in combinations of the following secondary antibodies and stains diluted in blocking solution: 1:500 Alexa Fluor 633 conjugated goat anti-mouse antibody , 1:500 Alexa Fluor 546 goat anti-rabbit antibody , 1:150 NeuroTrace 500/525 green fluorescent Nissl stain . Tissue was mounted on glass slides with 1–2 spacers  in Vectashield Hard Set Mounting Medium .Secondary-only controls for all antibodies were processed alongside regular staining to ascertain background fluorescence. No immunolabeling was observed in any tissues processed without the addition of primary antibody (not shown).The two anti-CALB2 antibodies utilized in the present study were generated against recombinant rat Calretinin . We confirmed antigen specificity of the antibodies by staining cryosections of mouse cerebellum (data not shown). In these controls we observed immunolabeling consistent with previous investigations , wherebyThe anti-TUBB3 antibody was produced against microtubules isolated from rat brain . This antibody has been widely used as a neuron specific marker and does not bind β-tubulin found in glia . Western blot analyses of the antibody against β3-tubulin demonstrated that this antibody recognized a doublet of bands in spiral ganglion extracts  consisteThe anti-MYOVI antibody was generated against amino acids 1049–1254 of porcine MYOVI . Anti-MYOVI is commonly used as a marker for hair cells including vestibular types I and II . ConsistConfocal images were captured on a Zeiss LSM 510 Meta confocal microscope implemented on an upright Axioplan 2 microscope using Zeiss LSM 510 software. The 488 nm (15% intensity), 543 nm (80–100% intensity) and 633 nm (10–20% intensity) laser lines were used for excitation. Bandpass filters of 505–530 nm, 560–615 nm, and a longpass filter 650 nm were used to capture separate emission channels. A Zeiss Plan-Neofluar 10X/0.3 NA objective was used to capture low-magnification images and high magnification images were obtained using a Zeiss Plan-Apochromat 63X/1.4 NA oil-immersion objective. Confocal stack micrographs were prepared for publication using Volocity software . Adobe Photoshop 7.0.1  was used to compile figures.Neurolucida . Hair cell quantification and sensory epithelia area measurements were completed using automated and semi-automated methods implemented within the Volocity software environment . Hair cell nuclei were differentiated from support cell nuclei based on position within the sensory epithelium, morphology, and chromatin condensation . Support cell nuclei were cuboidal, had compact chromatin, and formed a tightly packed monolayer over the basement membrane extending to the transitional epithelia at the ends of the sensory epithelia. In contrast, nuclei of hair cells were usually spherical, have less compact chromatin, and were located more toward the lumen compared to support cell nuclei.Parent axons and calyces of CALB2+ afferents were counted using the neuron tracing application in Hair cell nuclei within crista central zones and utricular peri- and extrastriola regions were included in density measurements. Cristae central zones were approximated as the area of sensory epithelia innervated by CALB2+ afferents. Two – four confocal stacks were sampled from each crista. Four utricle peristriola regions were systematically evaluated along the rostral to caudal line of polarity reversal (LPR), and encompassed the striola, defined as the region harboring CALB2+ calyces, and immediately adjacent areas. Two confocal stacks each from the lateral and medial striola were used to estimate hair cell densities in these regions. The borders of these image stacks constituted the counting frames for quantification. Nuclei contacting either of two adjacent sides of the counting frames were excluded from quantification, while nuclei contacting the other two sides were included in the counts. This estimation procedure was implemented to compensate for incomplete counting units transected by the counting frame. Support cell densities were quantified using similar methods to evaluate the treatment-induced alterations of the epithelia surface areas.2 of epithelium.We found that intraperilymphatic gentamicin administration resulted morphologic distortion in afferent calyces, and therefore established criteria on which to identify an “intact” calyx for quantification purposes. A CALB2+ calyx was counted if its height  extended to or above the apical-most level of the Nissl-stained hair cell nucleus. Density measures were computed as fractions of contralateral control density, and are also presented as CALB2+ calyces or axons per 100 μmThe areas of sensory epithelia used to determine densities of hair cells and CALB2+ parent axons and calyces were established by measuring the width along the support cell nuclei layer every 24 μm across the length of the cristae central zone (estimated by presence of CALB2+ parent axons) and utricle. The area was estimated as the product of the average width and the length.Figures 4A-v,vi) it was not possible to distinguish the epithelial origin of a given afferent. Furthermore, morphologic analyses were restricted to the central zones of the cristae. Direct associations between afferent discharge and morphologic characteristics of these crista regions were not made.It should be clarified that the characteristics of afferent electrophysiology and sensory epithelia morphology are intended to be representative of each metric within a given specimen. For most specimens, stimulus-evoked discharge modulation was lost or severely attenuated, and in all but a limited number of records  in two gentamicin-dosed specimens. This was accomplished through pneumatic injection of the label directly into the superior vestibular nerve in the vicinity of Scarpa’s ganglion . The label was allowed to incubate for 6 h, after which normal fixation and tissue harvesting procedures were completed.Figure 4) were computed as the reciprocal of ISI mean. Most perstimulus discharge records were obtained during 0.8 Hz, with the exception of Figure 4B for which the records were obtained during 0.4 Hz rotations.In this report most of the electrophysiologic data are represented as epochs of instantaneous discharge rates under the different gentamicin exposure conditions. The goal of these illustrations was to demonstrate the perstimulus discharge associated with the morphologic analyses. All instantaneous discharge rate waveforms were based upon spiketimes and derived from a Gaussian local rate filter using a 6.4 Hz corner frequency . Mean spregular afferents of the original utricular afferent dataset  was reported to be 54.2 ± 1.0 spikes⋅s-1 , and thAs specified above, coherence analyses were used to statistically verify stimulus-evoked discharge modulation. These measures were based upon multitaper spectral analyses , and werP and Q, KLD is computed from the formula (The distributions of spontaneous discharge characteristics were compared by computing Kullback–Leibler divergences (KLDs). KLD is a non-symmetrical measure of the difference between two probability distributions. For two discrete probability distributions  formula :P, while the compared distributions conformed to Q. A resampling strategy was implemented to test hypotheses that the KLD between two original distributions could have been derived from random sampling from the combined distributions . Software scripts used to compute KLD and manage the bootstrap resampling were written in the Igor environment . The resampling strategy was based upon one million resamples and subsequent KLD calculations. Probabilities to support the null hypothesis (specified above) were determined explicitly, except when the KLDs from the original distributions were outside the range of the distribution of KLDs generated from one million random resamples. In these cases, the probability is expressed as an inequality . Values less than 0.05 support rejection of the null hypothesis stated above, indicating that the two distributions could not be derived by random sampling from a dataset constituted from the combined distributions .Therefore, in the present study we established the convention that distributions representing semicircular canal afferents conformed to post hoc test and performed using GraphPad Prism 6.0 software . Data are presented as mean ± standard deviation (SD). Observed significance levels  are indicated in the figures with asterisks coded as follows: single asterisk (∗) reflects p < 0.05, double asterisks (∗∗) reflect p < 0.01, and triple asterisks (∗∗∗) reflect p < 0.001.Statistical comparisons of the different treatment groups were done by analysis of variance (single and multifactor ANOVA) with a Newman–Keuls Figures C. In each control condition similar simple and complex calyx arbor morphologies  to further illustrate similarities in CALB2+ calyx morphology among these control groups. These micrographs demonstrate that CALB2+ calyces in non-lesioned epithelia were strongly labeled from the parent axons to the fluted calyceal necks, which extended well above the nuclei of encapsulated hair cells.The cytoarchitecture of central zone epithelia from non-lesioned chinchilla cristae are presented first to provide the histologic perspective for the balance of the physiologic and morphologic correlates presented in this investigation. This was achieved through quantitative morphometry of normal (untreated), vehicle control (HBSS-infused) and contralesion control  cristae. In addition, these data provided the basis to determine whether the intraperilymphatic administration procedure induced histologic or physiologic changes that could have influenced our analyses independent of gentamicin’s effects. Maximum intensity projections of confocal image stacks from the central zones of CALB2-immunolabeled horizontal cristae, representing the three control groups, are shown in hologies  were obs2 area of the crista central zone), and compared (two-factor repeated measures ANOVA) across control condition  and crista identity . We found that the densities of each epithelial component were similar across control conditions as shown in Figures 1D,E and Tables 2 (p > 0.5). The similarities between vehicle controls and normal epithelia indicate that the chronic placement of the perilymph access port and HBSS infusion did not result in alterations of the morphologic parameters analyzed. Furthermore, despite the presence of a patent vestibular aqueduct in the chinchilla . While CALB2+ calyx densities  were similar in the posterior  and superior  cristae, mean CALB2+ calyx density of the horizontal cristae  was approximately 20% less than the vertical cristae (p < 0.001). Additionally, CALB2+ axon density (axons/100 μm2) was approximately 25% higher in posterior canal cristae  than in horizontal  and superior  cristae. In contrast to these differences in CALB2+ afferent innervation, mean central zone hair cell densities (HCs/100 μm2) were similar across all control cristae . These data suggest that the chinchilla superior cristae receive putative calyx-only afferents with a larger number of complex calyces compared to horizontal cristae. Additionally, the increased number of CALB2+ calyces in the posterior cristae, accompanied by an increase in parent axon density, reflects a greater number of putative calyx-only afferents.Though not a principal objective of this investigation, our analyses revealed differences in CALB2+ calyx and parent axon densities across cristae type . In addition, a population of non-lesioned afferents were derived from the vehicle-control specimens described above. These serve to test whether the surgical implantation of the perilymph port into the bony superior semicircular canal or the infusion of HBSS resulted in deleterious effects upon the physiology of superior and horizontal semicircular canal afferents. As discussed above, no differences were found in the morphology of CALB2+ calyces and hair cell densities in these specimens.The electrophysiology of non-lesioned chinchilla vestibular afferent neurons provides the background for evaluating the alterations concomitant with low-dose gentamicin administration. This background is provided primarily by our laboratory’s database of afferents recorded in normal, untreated specimens . In untreated specimens, the recording procedures utilized herein revealed afferent discharge characteristics similar to those previously reported . In addition, robust stimulus-evoked responses were also recorded from superior and horizontal semicircular canal afferents in Vehicle Control specimens (right). Like the Untreated specimens, semicircular canal afferents in these controls were clearly modulated in response to sinusoidal turntable rotation, indicating that perilymphatic port implantation and infusion of HBSS did not lead to deleterious outcomes with respect to afferent electrophysiologic measures. High measures of stimulus–response coherence (>0.85) were observed in these two afferent groups, even under conditions of modest discharge modulation (±5 spikes⋅s-1). Measures of stimulus–response coherence remained high in these controls.The balance of afferents probed at the recording site exhibited discharge that was clearly modulated during turntable rotations, as exemplified in pathotype, representing the different forms or “types” of pathology to specific components within the sensory epithelia. The goal of the present investigation was to refine production of the lesions so that unique pathotypes could be distinguished. Rather than strictly examine the lesions at the final mature stage, we envisioned that hair cell and afferent pathotypes might be distinguished if they exhibited differential temporal sensitivities to low-dose gentamicin. Therefore, we examined specimens at 0.5 and 1 months following gentamicin administration.Lesions to the peripheral vestibular receptors may involve hair cells, support cells, and afferent dendrites, inducing alterations in cellular function that may eventually lead to pathophysiologic changes in afferent discharge transmitted to the central nervous system. We refer to each general morphologic and physiologic component of the lesion as a Figure 3, and illustrate the heterogeneity in calyx morphology and associated physiology observed at these times. The morphologic and physiologic data shown in these figures were selected to be representative of the specimens, and direct associations between the afferent discharge records and the crista region highlighted in the micrographs cannot be made . Figures 3A,B show maximum intensity projection and orthogonal views (respectively) of the horizontal crista from a 0.5-month specimen exhibiting severe pathology to CALB2+ calyces. All CALB2+ calyces in this specimen were retracted, characterized by CALB2+ parent axons without calyces or drastically malformed partial calyces . The central zones of the three cristae from this specimen exhibited a mean decrease in hair cell density of approximately 24%. In the chinchilla crista central zone 60% of hair cells are type I  is shown. These records demonstrated that despite the histopathology exhibited by the crista epithelia  afferent discharge was clearly modulated in response to sinusoidal stimuli. Additionally, stimulus-evoked discharge modulation (approximately ±7–10 spikes⋅s-1) is superimposed upon spontaneous discharge (40–45 spikes⋅s-1), indicating that spontaneous discharge is preserved in these specimens that exhibited significant pathology. Coherence measures were comparable to that observed in untreated and vehicle control specimens .The discharge characteristics of afferents projecting to cristae of the preparation represented in Figures F, illustrating a unique histopathologic variant while the physiology of vestibular afferents demonstrated more severe functional compromise than shown in Figure 3C. Two of the three 1-month preparations exhibited complete calyx retraction, similar to that depicted by the micrographs in Figures 3A,B. The third preparation is shown in Figures 3D,E, illustrating CALB2+ calyces within the horizontal crista central zone that have lost the narrow fluted necks characteristic of untreated central zone calyces . The apical openings of most calyces in this specimen were widened. The inset micrograph, showing a volume reconstruction of the area enclosed by the dashed box , illustrates that the calyx opening was sufficiently large to enable visualization of the enclosed hair cell nuclei (yellow). Because these calyces extended above the hair cell nucleus, they were counted as intact calyces  despite the obvious morphologic anomaly.The histologic and physiologic results from a preparation examined at 1 month post-administration is shown in Figure 3F, which were recorded from the same preparation that yielded Figures 3D,E. These traces are shown to be representative of the unresponsive afferents recorded in this preparation, and they are not meant to be directly associated with the horizontal crista represented in the micrograph. The absence of stimulus-evoked discharge modulation precludes us from drawing the direct correlate. The stimulus–response coherence measures are shown for each trace, indicating the absence of discharge modulation to the stimulus . However, these afferents exhibited robust spontaneous discharge. This preparation is remarkable in that full calyx retraction had yet to dominate the crista landscape, yet afferent discharge reflected the severe response attenuation characteristic of other specimens evaluated at 1 month post-administration . This contrasts the clear stimulus-evoked discharge modulation in the 0.5 month specimen recorded in afferents projecting to cristae where central zone calyces have fully retracted . These observations supporting the conclusions that severe attenuation in stimulus-evoked afferent discharge modulation did not depend upon full calyx retraction, and that these two pathotypes are associated with independent mechanisms.The consistent electrophysiologic feature among all afferents recorded from 1-month post-administration preparations was the absence of responses to sinusoidal rotation. These are represented by the instantaneous discharge records shown in Figure 3, stimulus-evoked modulation was severely attenuated in all afferents recorded 1-month following gentamicin administration. Figure 4 demonstrates that this sensory dysfunction persists into post-administration times of 2 and 6 months. Detailed responses of afferents recorded from a 2-month preparation are shown in Figure 4A, illustrating paired perstimulus discharge from the same afferents during presentation of 0.8 Hz rotational sinusoids at 15  and 30°⋅s-1  peak stimulus velocities. The trajectories of these stimuli are illustrated by the bottom waveforms , and representative responses of horizontal  and superior  semicircular canal afferents from untreated specimens are shown at the top. The responses of three afferents from the same preparation are shown in Figures 4A-v–A-x, and illustrate the heterogeneity in the severe compromise found in this preparation. The histopathology of this horizontal crista is illustrated in Figure 7G, where complete loss of CALB2+ calyces was observed. Figures 4A-v,vi represent the discharge of a horizontal canal afferent exhibiting severely compromised responses to both 15 and 30°⋅s-1 stimulus intensities, but coherence analyses confirmed statistically verifiable responses . The perstimulus discharge records of the superior canal afferent  illustrated the case where coherence analysis indicated the absence of a response in the discharge at 15°⋅s-1, but the discharge during the 30°⋅s-1 peak velocity stimulus did evoke a verifiable response (p = 1.0⋅10-4). The perstimulus discharge of the third afferent , however, did not exhibit modulation by either stimulus magnitude (p = 0.11). These data demonstrate that severe response attenuation, and not solely response elimination, was a potential outcome of gentamicin administration at this post-administration time. In addition, these data illustrate the value in computing stimulus–response coherence as a firm metric to analyze these severely attenuated responses.As illustrated in Figure 4B. These data depict 12.5 s epochs of perstimulus discharge (in spikes⋅s-1) from 10 afferents recorded consecutively in a single preparation during presentation of 0.4 Hz sinusoidal rotations . For reference, representative response discharge recordings from superior and horizontal semicircular canal afferents in an untreated preparation at this stimulus intensity are shown in Figures 4B-ii,iii, respectively. The coherence values associated with these two responses were 0.96 and 0.99, with associated p-values of 1.4⋅10-10.and 1.0⋅10-18, respectively. Particularly notable is the high coherence of the superior canal afferent  despite the very low sensitivity and small modulation depth, indicating the coherence measures remain very high even in afferents for which the discharge modulation is extremely low.The physiologic ramifications of intraperilymphatic administration of 1 μg gentamicin at 6 months post-administration are shown in Figures 4B-iv–B-xiii, illustrating the perstimulus discharge during 0.4 Hz rotation . These data demonstrate three fundamental features of the electrophysiologic pathotype. First, coherence analyses verify the absence of rotational responses in all 10 perstimulus discharge records . While some of these afferents may have projected to the utricle , the probability that all ten afferents would project to the utricle is extremely low. Therefore, the absence of a response in all 10 afferents further verifies the severe response attenuation among afferents from treated labyrinths. Second, the compromise of response capabilities among semicircular canal afferents, first observed at 1 month post-administration, persisted through 6 months post-administration, which provided evidence indicating the vestibular sensory epithelia do not exhibit capabilities for spontaneous recovery by 6 months. Third, despite the severe response attenuation spontaneous discharge remained robust at this post-administration time, with some afferents exhibiting rates exceeding 80 spikes⋅s-1 . This demonstrated an absence of any progressive compromise in spontaneous discharge through the 6 month post-administration interval.The discharge characteristics representative of afferents recorded 6 months after receiving 1 μg intraperilymphatic gentamicin are represented in The principal finding from electrophysiologic recordings of vestibular afferents following intraperilymphatic administration of 1 μg gentamicin was the persistence of spontaneous discharge while modulation in response to head movement rotations was severely attenuated or lost. We tested the null hypothesis that spontaneous discharge characteristics of afferents recorded from gentamicin-treated preparations were similar to those of our laboratory’s database of semicircular canal afferents recorded from untreated preparations. We also conducted parallel analyses of previously published spontaneous discharge data recorded from normal (untreated) semicircular canal and utricular afferents . These lFigure 5A, against a backdrop of the characteristics obtained from our database of untreated semicircular canal afferents (black/gray symbols). The inset plot illustrates previously published spontaneous discharge data of semicircular canal and utricular afferents , histograms were prepared and are shown in Figure 5B (inset), in which the longer mean ISIs of gentamicin-treated afferents are visualized. The two distributions were compared by computing the KLD, after which a resampling strategy was implemented to test the hypothesis that the mean ISI distributions were derived from random sampling from a single underlying distribution. This probability was less than 1.0⋅10-6, supporting rejection of the null hypothesis. Furthermore, this result indicated that the distribution of mean ISIs from our sample of gentamicin-treated preparations corresponded to longer mean intervals compared to those of untreated semicircular canal afferents.The spontaneous discharge characteristics of afferents from gentamicin-treated preparations (green symbols) are shown in fferents , reconstFigure 5B represent the distributions of ISIs for semicircular canal and utricular afferents previously reported (p = 2⋅10-6). The difference in mean interval distributions between semicircular canal and gentamicin-treated afferents (KLD = 0.18) was more than twice that expected between semicircular canal and utricular afferents from the same preparations. This finding supports the conclusion that the difference in mean ISI distributions of untreated and gentamicin-treated preparations is much greater than that expected from canal-utricle differences.The histograms within the inset of reported . These dp = 2.6⋅10-4). However, similar resampling analyses showed that the CVs of previously published data from semicircular canal and utricular afferents were similar (p = 0.34). This latter analysis further supports the conclusion that the difference in CV distributions between gentamicin-treated and untreated afferents cannot be explained by a mis-categorization of rotationally insensitive utricular afferents as a characteristic of a gentamicin-treated pathotype.Spontaneous discharge coefficient of variation (CV) was also compared between untreated and gentamicin-treated distributions (KLD = 0.080), and between distributions of previously published data from semicircular canal and utricular afferents (KLD = 0.040). Through resampling analyses, we concluded that the CVs of gentamicin-treated afferents were greater than that of untreated afferents  and gentamicin-treated  specimens immunolabeled with anti-CALB2 (red) and anti-TUBB3 (green). In the contralesion control specimens , anti-TUBB3 immunolabeling (TUBB3+) extends into the calyx neck as was found for the anti-CALB2 labeling . The CALB2+ calyx defined type Ic hair cells . Control specimen hair cell density (hair cell nuclei per 100 μm2) of the peristriola region was estimated at 1.7 ± 0.15, and 2.5 ± 0.14 and 2.4 ± 0.12 for the lateral and medial extrastriola regions, respectively (N = 4). In comparison, hair cell densities of the gentamicin treated specimens were 1.1 ± 0.12 for the peristriola region, and 1.7 ± 0.51 and 1.5 ± 0.38 for the lateral and medial extrastriola regions . The relative densities computed from these absolute densities were similar to the central zone cristae at 6 months post-administration, which amounted to 64, 64, and 69% for the peristriola, medial extrastriola and lateral extrastriola (respectively).The measures of hair cell densities following low-dose gentamicin administration portray a different picture than that of the afferents. This is illustrated in 2) were similar for the 0.5-month , 1-month , 2-month , 6-month , and normal  specimens. These data support the conclusion that the alterations in epithelia morphology concomitant with gentamicin treatment could not account for the quantitative changes in hair cell or CALB2+ afferent densities.It has been previously reported that the thickness of the sensory epithelium  decreases as a consequence of the morphologic alterations induced by gentamicin . TherefoPrevious assessments of the distribution of hair cell types  indicated that type I hair cells represented approximately 60% of all hair cells within the central zones of chinchilla cristae . The losFigure 10, MYOVI immunoreactivity was robust in contralesion control hair cells that were tightly packed in the sensory epithelia . Hair cells with type I  and type II  morphologies were easily recognizable in optical orthogonal sections of the untreated specimens. Most type II hair cells exhibited the stereotypical morphology being broad around the more apically located nuclei and narrow and cylindrical at their bases. Type I hair cells exhibited the prototypical “chalice” morphology with a thickened plate atop the thin apical neck. This is further illustrated in the reconstructions of two type Ic hair cells shown in Figure 10D, highlighted for comparison to hair cells from gentamicin-treated epithelia. In this reconstruction, the CALB2+ calyces (red) were made translucent for visualization of the encapsulated MYOVI+ hair cells.Anti-MYOVI and anti-CALB2 immunohistochemistry was used to visualize hair cell morphology and the association with intact and remnant CALB2+ calyces in contralesion-control and gentamicin-treated specimens. As shown for superior semicircular canal cristae in Figures H, representing the central zone of a superior crista 6 months post-administration. While plenty of viable hair cells are indicated by robust anti-MYOVI immunolabeling, they were less densely packed reflecting hair cell loss . Hair cells exhibiting the stereotypical types I and II morphology were observed in few treated specimens (not shown); however, most hair cells lost distinguishing morphological characteristics as illustrated in the orthogonal optical section shown in Figure 10G. Some hair cells in damaged epithelia exhibited a “tear drop” morphology  while others exhibited a “barrel” morphology . The “tear drop” hair cells, although sharing similar morphological characteristics to type I hair cells, did not have the typical elongated neck of untreated type I hair cells. Some hair cells with ‘tear drop” morphologies were contacted by a retracted CALB2+ partial calyx as illustrated by the reconstruction in Figure 10H. In addition, partial calyces were observed to encapsulate only the bases of such “tear-drop” hair cells, inferring that hair cells with such morphologies are remnant type Ic hair cells.The morphologic changes in hair cells resulting from low-dose gentamicin administration is shown in Figures 11A,B show TRITC-labeled fibers (represented in green) projecting to the horizontal canal crista and utricle 6 months after gentamicin treatment, illustrating patent fibers projecting to the central zone and striola of these epithelia. In addition, these fine dendritic branches have morphological features representative of en passant and terminal boutons, characterized by varicosities along the trajectories and at the terminations of thin dendritic branches . Three-dimensional reconstructions of representative labeled fibers further illustrate the varicosities on small diameter branches  and show that large-intermediate diameter afferents with retracted calyces often were closely apposed to hair cell nuclei . Some of these fibers exhibited a bowl-shaped “hemi-calyx” ending at the base of the closely apposed hair cell .Although anti-TUBB3 immunohistochemistry revealed fine dendritic projections, it did not enable visualization of dendritic varicosities representing putative boutons of dimorphic afferent dendrites within the crista central zones or utricular striola , 1990. Tbranches , 1990. EFigure 12, the discussion of which will include the limitations and impacts of the outcomes of this experimental approach.Through the present investigation we implemented a method for direct intraperilymphatic gentamicin administration to achieve three principal objectives. First, we sought to produce a limited lesion among hair cells of the vestibular epithelia, thereby unmasking potential effects on the dendrites of primary afferent neurons . Second, we believed the refined strategy would limit the dosing variability and, therefore, achieve greater consistency in the outcome parameters. Our third objective was to elucidate the effects of lesioning both potential targets  on the discharge characteristics of afferent neurons. These results are summarized in the cartoon depicted in Figure 3 illustrates a case of extensive calyx loss even at 0.5 months. Some of the early calyx loss may be a downstream consequence of apoptosis . Therefore, the restricted lesion involving retraction of the calyx can be characterized as non-apoptotic. Data from the present study indicates that this lesion component may exhibit greater sensitivity, and perhaps have a greater impact, than hair cell loss in terms of the gentamicin-induced vestibular dysfunction or hypofunction.The administration of 1 μg gentamicin to the perilymph resulted in the loss of hair cells that was determined by comparing counts within the cristae central zones and utricular striolae from treated and contralateral control labyrinths. The mean hair cell counts ranged between 82.5 and 61.6% of hair cells in the contralateral labyrinths , corresponding to mean hair cell loss estimates between 17.5 and 38.4%. Since the fraction of type I hair cells found in the central zones and striolae of untreated epithelia are approximately 60% ,b, the mpoptosis  among th+/K+ATPases within calyceal membranes .While data of the present investigation do not provide direct insight into the pathophysiologic mechanisms of the lesions induced by low-dose gentamicin, they do provide a foundation upon which to build future investigations of the most labile components of vestibular dysfunction. In view of broad evidence indicating the deleterious action of gentamicin on mitochondria , and theembranes . GentamiFigure 10H).The present investigation did not enable resolution of the hair cell phenotypes that were lost upon gentamicin administration. The afferent calyx is the only morphologic characteristic that unambiguously identifies a type I hair cell. Other characteristics, such as general chalice morphology or nuclear location within the epithelia, are less definitive. Since gentamicin exposure, even at the low doses used in the present study, leads to calyx retraction and distortion of the epithelia that precludes the use of other characteristics (even if less reliable), it was not possible to estimate the contribution of hair cell phenotype to their total loss. Furthermore, the temporal profile of the loss suggests that there may be differential sensitivities whereby the most sensitive  are lost by 2 weeks, and another less sensitive group  that is lost over the succeeding 5.5 months. The proclivity with which type I hair cells have been shown to take up and retain gentamicin  suggestsThe goal of direct administration of gentamicin to the perilymph was to limit the variability in outcome measures to that associated with gentamicin pharmacology within vestibular epithelia and subsequent detection methods, eliminating variability associated with dosing and the quantity reaching the labyrinth. In only one preparation (0.5 months post-administration) was there no evidence of hair cell loss or calyx retraction, which we attribute to a failure of gentamicin administration. During port placement in this preparation, the subarcuate fossa adjacent to the bony superior canal was violated, which likely provided a pathway for misdirecting the infusate away from the labyrinth leading to the administration failure. Every other preparation resulted in hair cell loss and calyx pathology, and in preparations investigated at 1 month post-administration and longer severe attenuation of stimulus-evoked afferent discharge modulation was found. This supports the conclusion that these results provide a perspective of the inherent pathophysiologic variability of gentamicin administration that is independent of dosing variability. This includes: (1) hair cell and calyx loss that ensues within 2 weeks post-administration, exhibits modest variability through 1 month post-administration, but is highly stable after that time; (2) though severe response attenuation can be apparent within 2 weeks, some weak residual responsiveness can remain through 2 months; and (3) severe response attenuation persists through 6 months post-administration.Figures 6C,D), including those beyond the central zone and striolar boundaries. CALB2+ calyx pathology that was not representative of other calyces at shorter post-administration intervals would be indicative of a differential sensitivity to gentamicin, for which we are unaware of any evidence. Figures 6C,D demonstrate the absence of any such differential sensitivity at 6 months post-administration. As noted above, hair cell loss could not be distinguished by phenotype, including those designated type Ic  and Id hair cells   , providiFigures 4). The detection of stimulus-mediated responses was evaluated using stimulus–response coherence measures, which were determined from comparisons of frequency spectra contained in raw spike trains . Most coherence measures determined from the discharge of gentamicin-treated labyrinths were low by comparison, and the probabilities of chance correlation were typically high. Of course, it is always possible that increasing stimulus magnitude  would result in higher coherence between stimulus and response, as demonstrated by the superior canal afferent in Figures 4A-vii,viii. This reflects a threshold elevation due to the gentamicin treatment, which is not normally observed in mammalian afferents at comparable stimulus characteristics. Therefore, we interpret the low coherence measures among semicircular canal afferents from gentamicin-treated specimens, or the absence of stimulus–response coherence, as a severe attenuation in the capabilities for stimulus-evoked discharge modulation.In the present investigation evoked discharge modulation in vestibular afferent neurons was evaluated using stimulus characteristics for which robust responses were readily detected in untreated specimens . However, it is also important to note that Figure 4), but the absence of stimulus-evoked discharge was the predominant finding among most afferents recorded in the present study.A limitation of the present study concerns our inability to apply linear acceleration stimuli in the electrophysiologic evaluation of untreated or treated afferents. Figure 10H illustrates that the anatomical substrate for connectivity exists between viable hair cells and remnant parent axons even after calyces have retracted in putative calyx-only (CALB2+) afferents . Type II hair cells have also been shown to project onto parent axons through lateral extensions near the support cell layer  has become a clinical treatment for conditions of intractable vertigo associated with Mèniére’s syndrome  and as pWhile the present investigation utilizing normal animal subjects cannot provide direct insight into the etiology of Mèniére’s syndrome or other clinical conditions, it does provide important information regarding the pathophysiology of gentamicin-induced vestibular hypofunction. Direct intraperilymphatic administration eliminates potential dosing ambiguity associated with ITG, and while unlikely to be implemented as a clinical procedure it does provide insight into the outcomes that result under optimal administration conditions. Under the refined dosing conditions of the present investigation we found modest intrinsic variability within the first post-administration month across the limited number of subjects with respect to the morphologic  and physiologic  lesions. A critical topic in ITG therapy for intractable Mèniére’s syndrome concerns the relative level of induced hypofunction that optimizes the reduction in vertigo. Recent reviews of the clinical literature concerning ITG therapy are available  and summThe present study provides insight into the dose required to achieve the maximum level of hypofunction consistent with optimizing central compensation. That is, abrupt elimination of afferent input associated with vestibular schwannoma resection appears to be suboptimal in promoting central compensation, supporting the “prehab” strategy , 2009. WIt stands to reason that a critical goal of intraperilymphatic gentamicin is to provide permanent vertigo relief, and quite apart from the cases of treatment failure there is evidence that vertigo symptoms may return after initial relief following ITG treatment for Mèniére’s syndrome. While the use of small gentamicin doses unmasked a potentially critical effect on vestibular primary afferent dendrites, it also provides insight into other important aspects of vestibular pathophysiology. Certainly, establishment of precise ototoxicity protocols that render partial lesions to the vestibular epithelia are invaluable to the development of testing strategies for the early diagnosis of peripheral vestibular dysfunction. It also establishes a platform for the development of strategies for protection against ototoxicity resulting from systemic aminoglycoside therapy or chemotherapy. Furthermore, it must be recognized that the intratympanic administration (ITG) of gentamicin for the treatment of Mèniére’s disease is being conducted without an animal model exhibiting comparable level of pathology or dysfunction. That is, while partial dysfunction has been demonstrated in patients receiving ITG , a pathoDS and LH made substantial, direct, and intellectual contributions to the conception, execution, and analyses of the experimental work described, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the validation phase, individual genotyping of 44 variants showed three SNPs  remained associated with VA response at 6 months. SNP rs4910623 also associated with treatment response at 3 months (p = 1.5 × 10−3). Replication of these three SNPs in 376 patients revealed association of rs4910623 with poor VA response after 3 and 6 months of treatment . Meta-analysis of both cohorts (673 samples) confirmed association of rs4910623 with poor VA response after 3 months (p = 1.2 × 10−5) and 6 months (p = 9.3 × 10−6) of treatment in nAMD patients.Pooled DNA based GWAS to determine genetic association of SNPs with visual acuity (VA) outcome in anti-vascular endothelial growth factor (anti-VEGF) treated neovascular age-related macular degeneration (nAMD) patients. We performed pooled DNA based GWAS on 285 anti-VEGF treated nAMD patients using high density Illumina 4.3 M array. Primary outcome was change in VA in Early Treatment Diabetic Retinopathy Study (ETDRS) letters after 6 months of anti-VEGF treatment . GWAS analysis identified 44 SNPs of interest: 37 with strong evidence of association (p < 9 × 10 Age-related macular degeneration (AMD) is a common complex progressive neurodegenerative disease in the elderly, which can lead to irreversible severe vision loss45675It is well established that genetic factors are associated with risk of developing AMD81011The current study aimed to investigate associations with genetic variants in a hypothesis-free manner using a genome-wide association study (GWAS). We investigated whether genetic factors influencing ranibizumab treatment outcomes in nAMD patients could be identified through the initial use of a GWAS pooling strategy followed by a technical validation and subsequent replication in an independent cohort.The objective of this study was to investigate whether genetic variants could be identified that might influence the treatment outcome after anti-VEGF treatment in nAMD patients. The demographic characteristics of patients of the Melbourne discovery cohort and the replication cohort are shown in −8), SNPs in genes involved in drug resistance response (‘pharmagenes’)  and SNPs leading to a missense change in a coding region of a gene  (First phase results from the pooled GWAS are shown in a Manhattan plot in  × 10−4) .−5), in which the ‘G’ risk allele led to a worse response (OR = 2.58 [95% CI = 1.63–4.10]). While the C allele of both rs323085 and rs10158937  appeared protective  . This individual genotyping confirmed the 3 SNPs  to be significantly associated (p < 0.05) with response at 6 months of ranibizumab treatment after Bonferroni correction for 44 independent tests. SNP rs4910623 showed the highest significance (p = 5.7 × 10ctively) .−3, OR = 2.23 [95% CI = 1.36–3.66]).Since a loading dose of three monthly injections is prescribed to all patients, we determined if the effect of these three selected SNPs in treatment outcome could be seen at this time point. The G allele of rs4910623 showed an association with worse outcome at three months of treatment , consistent with the six months result. However rs323085 did not show significant association at three months . At the 3 month treatment time point all replication patients were included in the analysis (n  =  376) as they were all treated in a similar manner to the discovery cohort. The effect of this SNP on treatment response was also similar at this time point . SNPs rs323085 and rs10158937 were not associated with treatment response in the replication cohort  in the Melbourne discovery cohort was 71% and 72% for the non-responder group (3 and 6 months respectively) in comparison to 51% and 49% in the responders group  . A simil−5) and 6 months (p = 9.3 × 10−6) of treatment (only pro re nata patients from the replication cohort were included) indicating the influence of rs4910623 on outcome of ranibizumab treatment in AMD patients . As this group consisted of only 47 patients it was too small to draw any conclusive conclusions about whether this treatment protocol significantly affected the association of the G allele of rs4910623 with change in VA. In the meta-analysis we found significant association of rs4910623 patients with change in VA at both 3 and 6 months of treatment.We sought to identify genetic variants that influence the ranibizumab treatment response in AMD. Using a pooled DNA GWAS approach, we identified SNP rs4910623 in the promoter region of the 202 kb 3′ of LOC100287225and 42 kb at 3′ of LEPR gene respectively, did not remain associated with change in VA after replication. The inconsistency in the results for these two SNPs could indicate that they are false positive findings.Two other SNPs, rs323085 and rs10158937, located 2, using an optic disc diameter of 1.8 mm whereas the replication cohort was based on each patient’s optic disc area.The baseline characteristics of both the Melbourne discovery and replication cohort did not reveal significant association with change in VA except for the baseline VA at 3 and 6 months of treatment. Baseline VA is negatively associated with change in VA, thus lower baseline VA will lead to a greater VA gain after treatment and vice versa. Therefore our findings are in agreement with the ANCHOR and MARINA trials where higher VA at baseline was associated with a smaller gain in VA at 12 and 24 months of treatment, respectivelyOR52B4 gene. We also analysed the surrounding variants within its linkage disequilibrium (LD) block from the 1000 Genomes Project . A number of the identified SNPs in this LD block are predicted to alter transcriptional binding sites . Rhodopsin is also member of GPCR family and it functions to capture photons of light and trigger the intra-cellular photo-transduction cascade, forming the molecular basis of visionThe OR52B4 protein  consists of 314 amino acids (aa) and is a member of the G-coupled protein receptor (GPCR) family, which is located in the plasma membrane of the cell. Interestingly we find the conserved domain of Q8NGK2 OR52B4 gene in human eye tissue, we used online resource: “The Ocular Tissue Database”OR52B4 gene was present in the retina, optic nerve and choroid (olfr547 representing a homolog of the human OR52B4 gene (85% identity). Interestingly, olfr547 is a close relative of olfr78, which has recently been shown to function in regulation of blood pressure in smooth muscle cells222324OR10J5) was recently reported by Kim and colleaguesOR52B4 gene in angiogenesis needs to be elucidated and it has so far not previously been reported to be associated with pharmacogenetic response to AMD.To assess expression of the  choroid . The ideCFH gene has been associated with poor VA outcome in some studies but in other studies has been reported to show no association with VA. Similarly, inconsistent results of association were reported for ARMS2/HTRA1 11CFH, VEGF-A, HTRA1/ARMS2 Previous studies investigating genetic factors in anti-VEGF treatment outcome have assessed known AMD risk-associated genes and genes encoding components of the neovascularization pathway81011A1/ARMS2 . These fWe chose to use a pooled DNA GWAS strategy to investigate response to anti-VEGF treatment in AMD as this provided a cost-effective and efficient approach to identify genetic associations2330313234OR52B4 gene in models of angiogenesis, such studies could lead to the development of other therapies for AMD patients that can be used as an adjunct to existing treatment. This, in turn, offers the prospect of personalising treatment, based on genotype and opens up the route for exploring other drug treatment opportunities. In conclusion, we report for the first time a pooled DNA based GWAS on pharmacogenetic response to ranibizumab treatment in AMD that identifies association of variant rs4910623 in the OR52B4 gene. This finding was replicated in an independent replication cohort, suggesting that this gene may be involved in the response to anti-VEGF treatments in AMD patients of European decent. Finally, our data suggests that the SNP rs4910623 could be used as a diagnostic genetic marker before anti VEGF treatments for AMD.For the replication cohort, the number of injections at 6 month of treatment was not available, however it is interesting to note that a variation in treatment regime through change in number of injections between 3 and 6 months does not appear to ameliorate the genetic effect seen in the current study. However, after individual genotyping, replication and meta-analysis, SNP rs4910623 remained associated with worse functional outcome (change in VA) after the anti-VEGF treatment in AMD. Currently there is much interest in the role of anatomical features such as fluid clearance as a measure of treatment response. However, it was not considered in this analysis but this would be useful to examine in the future as to whether this SNP also affects fluid clearance and central macular thickness (CMT) on OCT following anti-VEGF treatment. To our knowledge, SNP rs4910623 and the 2 other SNPs associated with good response in the discovery phase have not previously been reported as associated with anti-VEGF treatment outcome in any type of cancer, thus it will be interesting to investigate the role of these SNPs in cancer following anti-VEGF treatment. Furthermore, much work needs to be undertaken to examine the biological basis of this phenomenon as well as the functional role of th revision). Written informed consent was obtained for all study participants before participation. Patients were included in the study when they were >50 years of age, presented with an active sub-foveal CNV secondary to AMD, and received intravitreal anti-VEGF injections. Neovascularization was confirmed by fundus photography , fundus fluorescein angiography (FFA) using IMAGEnet 2000 , and optical coherence tomography (OCT) with Stratus OCT version 5.0.1  or Cirrus HD-OCT version 6.0.0.599 (Carl Zeiss Meditec). Exclusion criteria were nAMD associated with non-AMD conditions such as degenerative myopia, angioid streaks and hereditary retinal disorders. In addition bilateral nAMD patients who had different VA response between both eyes and treatment with either laser photocoagulation or photodynamic therapy before anti-VEGF treatment as previously reported were also excludedThe study was approved by the Human Research and Ethics Committee of the Royal Victorian Eye and Ear Hospital (RVEEH) and performed in accordance to the tenets of the Declaration of Helsinki (7VA was measured using an Early Treatment Diabetic Retinopathy Study (ETDRS) chart at 4-metre distance at each visit. The majority of nAMD patients were treated with monthly ranibizumab injections  for 3 months of initial dosing except for 27 (9.0%) patients who first received a single injection of bevacizumab  who then went on to receive normal dosing using ranibizumab. After 3 monthly injections, pro re nata treatment regimen were adopted for follow-up injections, that was based on evaluation by a retinal specialist, who considered loss of VA ≥ 5 EDTRS letters, the presence of fluid on an OCT scan or presence of chronic or new retinal haemorrhage as criteria for treatment. Data on demographics and clinical history (including VA and treatment regimens) were collected for all participating patients.Ethical approval for the replication cohort was obtained from the local ethic committees of the Radboud university medical center, University of Cologne, and McGill University Health Centre. Written informed consent was acquired from all participants.The replication cohort consisted of 376 treatment naïve patients of European descent with choroidal neovascularization secondary to AMD. Patients were diagnosed by retinal specialists based on ophthalmic examination, spectral-domain OCT  or fluorescein angiography (FA) . All included patients were over the age of 50 years, had not undergone any previous ophthalmic surgery, except for cataract removal, and did not have other retinal disorders besides AMD.pro re nata regimen at the clinics of Nijmegen and Cologne and on a treat-and-extend regimen at the clinic of Montreal. In Nijmegen and Cologne (pro re nata regimen), in cases of persistence or recurrence of choroidal neovascularization, which was defined as loss of VA of ≥5 ETDRS letters, leakage seen on FA, fluid seen by OCT, or new macular hemorrhage or fluid, three consecutive monthly ranibizumab injections were administered. VA was assessed before treatment and after the three loading injections for all patients. VA was also recorded after 6 months of treatment for 262 patients. For 303 patients, Snellen VA measurements were recorded retrospectively and 73 patients were followed up prospectively using ETDRS VA. Baseline variables were collected using questionnaires or retrieved from the patient files. One eye was selected per patient; if both eyes received treatment, the first eye treated was selected and if treatment started simultaneously, the study eye was chosen randomly.A total of 144 patients were treated at the Department of Ophthalmology of the Radboud university medical centre, Nijmegen, the Netherlands and 182 at the University of Cologne, Germany. These patients were included in the European Genetic Database (EUGENDA), a multicenter database for the clinical and molecular analysis of AMD. The remaining 50 patients were treated at the McGill University Health Center, Montreal, Canada. All patients received the loading dose of three consecutive monthly intravitreal injections of 0.5 mg ranibizumab . Afterwards, they were followed up monthly and treated on a et al.et al.In the first phase of the study, a GWAS using a DNA pooling approach was conducted  and performed as previously describedIn the 2nd phase of the study were analysed in an independent ranibizumab-treated AMD cohort of European descent. DNA was extracted and quantified in Nijmegen, the Netherlands. Subsequently, DNA samples of a total of 376 AMD patients were genotyped in Melbourne using the MassArray platform  as previously describedIn the third phase of the study, SNPs that showed association (p < 0.05 after correction for multiple testing) in the 2Linear regression was undertaken to assess the role of non-genetic factors , including age, sex, smoking status, CNV size, lesion type, number of injections and baseline VA, on the dependent variable “change in VA” at 3 and 6 months respectively.The GWAS of pooled DNA was undertaken at the Queensland Institute of Medical Research (QIMR) as previously described37p < 0.05 following Bonferroni correction for multiple testing. For the analyses, the statistical software Plink V1.07 (http://pngu.mgh.harvard.edu/~purcell/plink/) and SPSS IBM SPSS Statistics for Windows, version 20.0  were used. To prevent any possible confounding, the analyses performed in the both the Melbourne discovery and the replication cohort were corrected for baseline VA.For individual genotyping data, logistic regression was used to assess differences in allele frequency between the responder and non-responder groups (defined in the same manner as for the GWAS) and adjusted for baseline VA in the discovery cohort and baseline VA and age at first injection in the replication cohort. In this replication cohort, different treatment regimens were administered after the 3 initial monthly injections. Therefore, we stratified the analysis for the replication cohort into two groups; pro-re-nata and treat-and-extend. These groups consisted of 215 pro re nata (Nijmegen and Cologne) and 47 treat-and-extend  patients respectively. Results are reported as odds ratios (OR), 95% confidence intervals (95% CI) and p value with statistical significance being defined as Meta-analysis of the discovery and replication cohorts was performed using Metal softwareHow to cite this article: Riaz, M. et al. GWAS study using DNA pooling strategy identifies association of variant rs4910623 in OR52B4 gene with anti-VEGF treatment response in age-related macular degeneration. Sci. Rep.6, 37924; doi: 10.1038/srep37924 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


NAV1, IGF2, GNAS, and CRTC1. As an initial validation of these findings, we tested the significance of an overlap of these data with anxiety-related differentially methylated loci that we previously reported from a key neural circuit of anxiety  in young monkeys and found a significant overlap  of anxiety-related differentially methylated genes, including GNAS, SYN3, and JAG2. Finally, sequence motif predictions of all the human differentially methylated regions indicated an enrichment of five transcription factor binding motifs, suggesting that DNA methylation may regulate gene expression by mediating transcription factor binding of these transcripts. Together, these data demonstrate environmentally sensitive factors that may underlie the development of human anxiety.The etiology of individual differences in human anxiousness is complex and includes contributions from genetic, epigenetic  and environmental factors. Past genomic approaches have been limited in their ability to detect human anxiety-related differences in these factors. To overcome these limitations, we employed both a multi-dimensional characterization method, to select monozygotic twin pairs discordant for anxiety, and whole genome DNA methylation sequencing. This approach revealed 230 anxiety-related differentially methylated loci that were annotated to 183 genes, including several known stress-related genes such as  Trait-like anxiety in humans and non-human primates is associated with stable individual differences in hypothalamic–pituitary–adrenal (HPA) axis activation and amygdala function3. HPA activation results in the release of cortisol, and increased cortisol concentrations in children and adolescents can be linked to inhibited behaviors and anxiety that often persist throughout life5. Additionally, a loss of the ‘natural’ circadian decline in afternoon/evening cortisol levels has been correlated with shyness and later alterations in behavior, including internalizing problems7, suggesting that late-in-the day cortisol levels in children and adolescents may be an index of early life and current stress exposure as well as altered behaviors. High afternoon cortisol levels in childhood are also negatively correlated with amygdala–prefrontal cortex connectivity in adolescents and adults, indicating that a disruption in amygdala function is related to trait-like anxiety12. In fact, anxiety prone individuals show greater amygdala activation during emotion processing tasks, further supporting a central role of the amygdala in processing of fearful stimuli15. Moreover, lesions in the central nucleus of the amygdala of non-human primates results in decreased adrenocorticortropic hormone (ACTH) concentrations before and after stressful conditions16. Finally, higher and prolonged amygdala metabolism following a stressful challenge results in increased anxiety-like behaviors  in young rhesus monkeys17, suggesting that the timing of amygdala activation and deactivation, in both humans and rhesus monkeys, is associated with trait-like anxiety.Anxiety is frequently characterized by a negative affective response that is associated with the anticipation of encountering a potential threat18. Studies using adult post-mortem brain tissue support a role for DNA methylation ) in the development of anxiety, bipolar disorder, schizophrenia, and major depressive disorder24. Our recent study in young monkeys, as well as studies in humans, identified differentially methylated genes that are implicated as risk factors for anxiety and depressive disorders26. Thus, these studies support the hypothesis that DNA methylation may have an important role in the risk to develop trait-like anxiety. However, these studies have relied heavily on the ability to access brain tissue. Focusing studies on anxiety-related DNA methylation profiles in blood has the potential to provide tools that could be clinically utilized to improve diagnostic and treatment strategies.Genetic data suggest that common anxiety disorders like generalized and social anxiety disorders are ~20–40% heritable and that environmental factors—potentially including epigenetic modifications—likely account for much of the remaining variability29. In addition, a substantial portion of the individual variability in anxiety level is due to variations in non-genetic factors30. The monozygotic (MZ) twin difference design9. Therefore, we anticipated finding an environmentally mediated relationship between cortisol levels and anxiety symptoms in a large birth-based sample of MZ twin pairs. Under the assumption that the MZ co-twin with higher childhood afternoon basal cortisol levels and protracted amygdala response would have more adolescent anxiety symptoms than their co-twin with lower cortisol and amygdala activation, we selected three MZ twin pairs from Burghy and colleagues that were maximally discordant on these phenotypes9. In these individuals, whole genome DNA methylation levels were profiled to identify anxiety-related DNA methylation in human blood.Twin studies showed that afternoon cortisol levels and amygdala volume are strongly influenced by environmental  factors31 were invited to participate in a research panel, with 74% agreeing. At age 7 years  twins were screened via telephone interview with the primary caregiver (>95% mothers) with the goal of mildly enriching the sample for risk for behavior problems . This mild selection was intended to introduce more variance in symptoms than an entirely unselected sample would show. Shortly after screening, selected participants  took part in a multi-informant, multi-method assessment at age 8 years . Participants were invited to enroll in two follow-up studies at age 13  and age 15 . Informed consent  was obtained for all assessments, and participants received monetary compensation. University of Wisconsin–Madison Institutional Review Boards approved all procedures. All methods were carried out in accordance with the approved guidelines.Between 1997 and 2002, families identified from Wisconsin state birth recordsA subset of MZ twins were also selected for a neuroimaging study if one or both twins experienced chronic anxiety ; 13.5% of participants met criteria. Pairs were considered discordant if one twin experienced chronic anxiety and the co-twin did not meet criteria for any anxiety disorder at any assessment occasion (5%); pairs were concordant if both co-twins experienced chronic anxiety (6%). The majority of twin pairs were neither concordant nor discordant. Twenty-four pairs participated in the neuroimaging study .31. On average the mother’s education corresponded to some technical or college experience. Median family income falls in the $50,000–$60,000 range.Characteristics based on demographics collected at age 8 years are shown in Supplementary Table 32 and Diagnostic Predictive Scales33 to parents and adolescent offspring. Parents completed the Health and Behavior Questionnaire7 (HBQ) at all waves of data collection. Offspring completed the HBQ during both adolescent assessments. We constructed a general anxiety composite consisting of standardized DISC generalized anxiety disorder symptom counts averaged with standardized HBQ over anxiousness scores, and we constructed a social anxiety composite in the same manner. Parent (age 8 years) and offspring (ages 13 and 15 years) scores were kept separate.Trained interviewers administered the Diagnostic Interview Schedule for Children (DISC)34) to both parents and adolescent offspring during the imaging visit. The K-SADS provides past and current diagnoses and past and current subclinical symptoms.Clinicians administered the Schedule for Affective Disorders and Schizophrenia for School-Age Children , and 30 min prior to bedtime on three consecutive days. Cortisol was assessed in duplicate with a salivary enzyme immunoassay kit . Repeat assays were performed on any samples not meeting quality control requirements. Families were assayed across one or two batches. Log-transformed afternoon cortisol values were regressed on time since waking before averaging over the three collection days.3 voxels) were acquired axially with an isotropic 3D Bravo sequence . T2*-weighted gradient-echo echo-planar pulse sequence images were collected during with TE = 25 ms, TR = 2000 ms and flip angle = 60°. For full data reduction and processing, see Burgy et al. 20169.At a mean age 15 years, structural and functional images were collected on a 3 T MRI scanner  with an 8-channel RF head coil array. T1-weighted structural images  divided over 5 blocks. A white fixation cross was displayed in the center of a black screen (1 s), followed by a picture (4 s). Participants were instructed to indicate the valence of each image via button press. Following picture offset, a second fixation screen was presented. Two thirds of trials included a neutral face condition . After the offset of face images, the fixation cross was represented with an inter-trial interval that varied from 5.5 to 17.6 s (M = 8.89 s), providing sufficient variation to estimate the evoked BOLD response function. The remaining trials had no face presentation. The presentation of the faces allowed a behavioral measure of post-image recovery. Faces following negative images were rated as less likable than faces following neutral images or novel faces9.Participants completed a passive picture-viewing task to index emotional reactivity and recovery  from baseline in each epoch. To unconfound recovery from reactivity, initial PSC in the reactivity epoch was regressed onto PSC estimates of modulated recovery. Face trials were calculated with a double-subtraction of face and no face trials: —. These contrasts were warped to MNI space, smoothed (FWHM = 6 mm), and intrapair contrasts were calculated. Both voxelwise and amygdala seed data were examined .To examine reactivity and recovery effects, no face and neutral face trials were separated into two 6 s epochs beginning at IAPS image onset, with the first epoch representing the initial response to the image (reactivity), and the second representing the neural recovery to the image and response to the face presentation, where applicable and as we previously reportedWhole blood was collected from each participant in a BD vacutainer CPT cell preparation tube with sodium heparin (cat # 362753). The peripheral blood mononuclear cells were isolated and genomic DNA was extracted using Promega wizard genomic DNA purification kit (cat #A1120), following the manufacturer’s protocol.Genome-wide methylation data were generated by WuXi NextCode for each sample using whole-genome sequencing technologies from Illumina (HiSeq X). Briefly, genomic DNA (200 ng) was randomly fragmented, end-repaired, and ligated to NEBNext Methylated Adapter for Illumina following the manufacturer’s protocol (Illumina). Adapter-ligated DNA fragments, ranging from 200 to 400 base pairs (bp), were purified by Sample Purification Beads (Illumina) and then treated with sodium bisulfite (ZymoResearch EZ DNA methylation gold kit), which converts unmethylated cytosines to uracil and leaves methylated cytosines unchanged. Libraries of converted DNA fragments were then amplified using KAPA HiFi Hot Start Uracil + Ready Mix (KAPA Biosystems KM2801), Index Primer for Illumina and Universal PCR Primer for Illumina (NEB E7336A), and amplicons were purified by Sample Purification Beads (Illumina) and sequenced on a Next-Generation sequencer (Illumina HiSeq X). This approach yielded 650–800 million 150 bp-reads for each library. Image processing and sequence extraction used the standard Illumina Pipeline.36 and bismark (version 0.17.0)39. The average number of raw reads for each sample (N = 6) was 404 million reads giving an average genomic coverage of 20.23× (median genomic coverage 19.53×). The sequence reads were filtered for low quality and adaptor sequences were removed. The cleaned reads were then mapped to the human reference genome (hg38) and an average of 283.3 million uniquely mapped reads were obtained for each sample, giving an average coverage of 14.16× (median coverage 13.86×). Sequence reads from both DNA strands (forward and reverse) were combined to determine the DNA methylation level at all CpG dinucleotides (~25.3 million). Differentially methylated regions (DMRs) were identified using the DSS-single analysis method41, which was selected because it incorporates the read depth into the DMR analysis and relies on smoothing so that neighborhood CpGs can be viewed as pseudo-replicates and dispersion can be estimated across an entire genomic window. All default settings were used in the DSS package (including a smoothing span of 500 bp). Notably, sex chromosomes were excluded from DMR analysis because the samples included both male and female pairs. DMRs were identified using DSS, and limiting DMRs to those having a minimum of 5 CpG dinucleotides and a difference in mean methylation of 10% between the two groups.Quality control, mapping, and extraction of methylation information from the whole genome sequence data were done using bowtie2P-values for over-representation of a set of genes to specific Gene Ontology terms. It uses a hypergeometric test to calculate the P-values, given a set of genes and a gene universe .Gene Ontology (GO) analysis was conducted in R using the ‘topGO’ package. This package calculates the 42. Finally, enriched motifs were placed in SpaMo to predict transcription factors that may putatively bind to these discovered motifs43.To identify sequence motif enrichments, the center coordinate was first identified for each DMR, then extended ±500 bp around this center coordinate. Sequences were next obtained for these coordinates using the human genome (hg38) and placed in the Discriminative Regular Expression Motif Elicitation (DREME) tool to identify enriched short, ungapped motifs from these sequences relative to random sequences used as a background44. For MZ twins, a low ICC indicates that the trait has low heritability and large non-shared environmental influences. The ICCs for GAD and social phobia dropped substantially from a high of .56 at age 8  to a low of .40 at age 15 , social phobia, HPA activity (cortisol concentration), and amygdala function, we computed intraclass correlations (ICCs)) to quantify the degree of within-pair similarity between MZ cotwins over a seven-year periodP-value < 0.001; N = 299 pairs), we previously showed that afternoon basal cortisol is strongly influenced by shared environment, and the remaining variance in this early cortisol measure can be attributed to non-shared environmental influences28.Despite finding that afternoon cortisol concentrations at age 8 showed high intrapair similarity , our measure of interest, amygdala recovery, showed low and statistically non-significant twin pair similarity . These data suggest that the time course for recovery  is particularly sensitive to a non-shared environment  at age 15 was moderately familial  at ages 13 and 15 years on intrapair differences in afternoon cortisol at age eight. The cortisol differences predicted co-twin differences in general anxiety at age 13 years  and social phobia at age 15 years . Together with our previous findings, that showed a positive relationship between intrapair differences in childhood afternoon cortisol levels and intrapair differences in protracted amygdala activation in adolescence9, these data suggest that the MZ difference design could be used to select uniquely discordant twins based on significant differences in these measures, which might provide an opportunity to examine possible environmentally sensitive molecular changes  related to twin discordance in biological systems related to anxiety.We next employed the MZ twin difference design to determine if intrapair differences in cortisol predict intrapair ; Fig. Using this approach, we selected three twin pairs that were the most discordant along two axes—cortisol levels at age eight and amygdala-modulated recovery at age 15—for whole-genome analysis of DNA methylation. These time points were chosen because all three selected pairs were clearly discordant for HPA activity at age 8 years Fig. , but theN = 23,444,312 CpG dinucleotides) and this final dataset revealed a bimodal distribution of DNA methylation in human blood cells, with the majority (>60%) of CpGs being more than 60% methylated . This approach generated an average of ~404 million raw sequence reads for each sample and, after filtering for quality, an average of 283.3 million sequence reads uniquely mapped to the human reference genome (hg38), giving an average genomic coverage of >14× . This analytical approach, which limited positive results to differentially methylated regions (DMRs) that have a minimum of 5 adjacent CpG dinucleotides and a minimum mean intrapair methylation difference of 10% across the three twin pairs, revealed a total of 230 anxiety-related differentially methylated loci. Anxiety-related increases in methylation were classified as hyper-DMRs and anxiety-related decreases in methylation were classified as hypo-DMRs. A total of 176 hyper- and 54 hypo-DMRs were identified and these loci were distributed across all the autosomes  of anxiety-related differentially methylated genes, including GNAS, SYN3, and JAG2 . Together, these data suggest a commonality in the molecular underpinnings of anxiety in monkeys and humans, providing validation of our multi-species approach and promising candidate genes.As an initial validation of these findings, we tested for significance in an overlap with anxiety-related differentially methylated loci that we previously reported in the central nucleus of the amygdala of young monkeysG2 Table . Notably42. This analysis yielded five TF binding sequences that were significantly enriched within the DMR sequences  and general development  binding sequences, using the Discriminative Regular Expression Motif Elicitation (DREME) suite software package ces Fig. . These T Fig. TCF and gene17. Epigenetic modifications are one mechanism by which experiential factors influence anxious behaviors. Our goal was not to examine a representative sample, but to maximize MZ intrapair differences to determine the potential for epigenetic modifications in human blood related to human anxiety. We identified 230 loci with anxiety-related DNA methylation differences in blood of MZ twins that were selected based on discordance in cortisol and MRI measures related to anxiety. The power of whole genome sequence data was critical to detection, as mean methylation levels are strongly correlated across the genome and statistical power was increased by ‘borrowing’ strength across adjacent measurements54.Many twin studies have investigated the extent to which anxiety is influenced by latent environmental factorsGNAS, SYN3, and JAG2, for future mechanistic studies in the nonhuman primate model.Although using relevant tissue is important for the biological interpretation of epigenome-wide association studies, obtaining such samples can be challenging for human studies of brain-related disorders. The significant overlap with the previously reported anxious temperament-associated differentially methylated loci from a key component of the neural circuit underlying primate anxiety provides a corroboration of the genes found here and suggests candidate genes, including 55. We might have expected some overlap under the hypothesis that sequence variants in the same genes could have similar biological effects to changes in DNA methylation. Nonetheless, several genes that we detected with DMRs have well-known links to neurodevelopmental disorders. For example, the stimulatory G-protein alpha subunit (GNAS) is located in a complex imprinted locus whose gene products are involved in early postnatal adaptations and neuroendocrine functions56. Maternal stress during pregnancy is linked with differential methylation on GNAS and insulin-like growth factor 2 (IGF2) gene57, which also was found here with an anxiety-related DMR. Synapsin 3 (SYN3) is a member of a gene family that encodes neuronal phosphoproteins that associate with the cytoplasmic surface of synaptic vesicles47. SYN3 has a role in synaptogenesis and the modulation of neurotransmitter release, leading to its link to several neuropsychiatric conditions, including bipolar disorder, autism, schizophrenia and epilepsy59. We previously reported anxiety-related differential methylation of JAG1 and JAG2 in the central nucleus of the amygdala of young monkeys;26 here we find anxiety-related differential methylation of JAG2 in humans. These genes are biologically relevant candidate genes contributing to anxiety because they are NOTCH receptor genes that play a critical role in brain development, adult synaptic plasticity, and memory formation. Finally, these findings also revealed potentially novel genes contributing to anxiety  and 87 DMRs that were not associated with any genes, suggesting that they may reside in non-coding regions of the genome and warrant a deeper investigation.A cautionary note is that comparison of these genes to those from a meta-analysis of genome-wide association studies for anxiety disorders did not find an overlap of genesTCF3) is a neurodevelopmental transcription factor that directly enhances the expression of HES1, which regulates the development of the central nervous system through NOTCH signaling50. The Retinoid-Related Orphan Receptor-Alpha (RORA) is a key regulator of embryonic development, cellular differentiation, immunity, and circadian rhythm62. RORA is linked with an increased risk for several neuropsychiatric conditions, including autism spectrum, bipolar disorder, schizophrenia, depression, and fear-related psychopathology64. Finally, the dysregulation of SMAD genes reportedly affect oligodendrogenesis49, axon development and regeneration, growth and maintenance of midbrain dopaminergic neurons65. Alterations in SMAD-related molecular cascades are reported for various psychiatric conditions70.Several transcription factors that recognize significantly enriched sequence motifs within the DMRs have known roles in neurogenesis and neurological activities. Transcription factor 3 (TCF3 and a member of the Kruppel-like factor (KLF) family (KLF-6), contain DMRs, suggesting that DNA methylation may also disrupt the expression of transcription factors. The involvement of DNA methylation in the regulation of KLF family members is important because they are required for late phase neuronal maturation in the developing dentate gyrus during adult hippocampal neurogenesis71. Murine studies find associations with Klf-9 and anxiety, and human studies link Klf-11 with chronic stress and depressive disorders72. While these data support previous reports76 of differential methylation associated with transcription factor binding sites, it is notable that here, and in these reports, the majority of transcription factor binding sites do not contain CpG dinucleotides directly in the transcription factor binding motif. Recent data suggest that TF complexes may contain DNA methyltransferases, implying that some TFs may function to bring DNA methyltransferases to genomic regions of interest77. Clearly, further functional studies are needed to definitively determine the role of differential methylation within transcription factor binding sites related to anxiety.In addition, several of the genes that encode the transcription factors, such as 78. Nonetheless, sensitivity to daily stressors, as embodied by environmentally mediated variability in basal cortisol levels, may be an early, subtle indicator of vulnerability to later anxiety symptoms. Although the twin pairs were not selected on measures of anxiety, all three were discordant in the same direction such that the co-twin with higher cortisol and longer amygdala recovery was also the more anxious co-twin. At the individual level, HPA function at age 8 years was not related to anxiety longitudinally or concurrently in the full sample. The association between HPA function and anxious behavior was only revealed after controlling for unknown genetic and environmental confounds shared by twins reared together. Finally, it is notable that the selected twins reported fluctuations in the level of anxiety between the imaging and blood draw visits. While there are a number of reasons that a diagnosis could change between visits, including reluctance to disclose symptoms, effective treatment of prior symptoms, and spontaneous remediation, the changes in DNA methylation that are linked to adolescent anxiety status appears to be stable and may be useful for long-term diagnostics and treatment strategies.Although the twin pairs were clearly discordant for HPA activity at age 8 years, they did not become discordant for behavioral measures of anxiety until mid-adolescence. In fact, the selected co-twins, and the sample in general, are remarkably similar on anxiety measures at age 13 years, and start to diverge only 2 years later. This finding may not be surprising given the increased prevalence for anxious behaviors that coincide with the onset of pubertyN = 1; Supplementary Table It should be noted that the original anxiety-related changes in DNA methylation were found in young monkeys (<2 years) that had never received anxiety-related medications, which are commonly administered to humans with anxiety disorders. Together, these study design parameters improve the chances that the identified anxiety-related changes in DNA methylation have etiological relevance rather than being a consequence of treatment. Because none of the twin pairs were on any medications , which can be found under the Gene Series: TBD.Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplemental Table 1Supplemental Table 2Supplemental Table 3