Reprogramming to recover youthful epigenetic information and restore vision.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.

LCM-seq reveals unique transcriptional adaptation mechanisms of resistant neurons and identifies protective pathways in spinal muscular atrophy.

Somatic motor neurons are selectively vulnerable in spinal muscular atrophy (SMA), which is caused by a deficiency of the ubiquitously expressed survival of motor neuron protein. However, some motor neuron groups, including oculomotor and trochlear (ocular), which innervate eye muscles, are for unknown reasons spared. To reveal mechanisms of vulnerability and resistance in SMA, we investigate the transcriptional dynamics in discrete neuronal populations using laser capture microdissection coupled with RNA sequencing (LCM-seq). Using gene correlation network analysis, we reveal a TRP53-mediated stress response that is intrinsic to all somatic motor neurons independent of their vulnerability, but absent in relatively resistant red nucleus and visceral motor neurons. However, the temporal and spatial expression analysis across neuron types shows that the majority of SMA-induced modulations are cell type-specific. Using Gene Ontology and protein network analyses, we show that ocular motor neurons present unique disease-adaptation mechanisms that could explain their resilience. Specifically, ocular motor neurons up-regulate (1) Syt1, Syt5, and Cplx2, which modulate neurotransmitter release; (2) the neuronal survival factors Gdf15, Chl1, and Lif; (3) Aldh4, that protects cells from oxidative stress; and (4) the caspase inhibitor Pak4. Finally, we show that GDF15 can rescue vulnerable human spinal motor neurons from degeneration. This confirms that adaptation mechanisms identified in resilient neurons can be used to reduce susceptibility of vulnerable neurons. In conclusion, this in-depth longitudinal transcriptomics analysis in SMA reveals novel cell type-specific changes that, alone and combined, present compelling targets, including Gdf15, for future gene therapy studies aimed toward preserving vulnerable motor neurons.

The after-effect of noisy galvanic vestibular stimulation on postural control in young people: A randomized controlled trial.

Noisy galvanic vestibular stimulation (nGVS) enhances the vestibular system. The center of pressure (COP) sway has been shown to decrease during nGVS, but the after-effect of nGVS remains unclear. The aim of this study is to elucidate the after-effect of nGVS on COP sway. We randomly assigned 26 participants to either control (sham stimulation) or nGVS groups. All participants were measured for COP sway while standing with open eyes at baseline, during stimulation, and after stimulation. In the nGVS group, sway path length, mediolateral mean velocity, and anteroposterior mean velocity decreased both during stimulation and after stimulation compared with baseline. Conversely, no significant difference in COP sway was detected in the control group. There was a correlation between the stimulation effect and the after-effect in the nGVS group, indicating that nGVS is effective for people with high baseline COP sway.

Optimizing the ICA-based removal of ocular EEG artifacts from free viewing experiments.

Combining EEG with eye-tracking is a promising approach to study neural correlates of natural vision, but the resulting recordings are also heavily contaminated by activity of the eye balls, eye lids, and extraocular muscles. While Independent Component Analysis (ICA) is commonly used to suppress these ocular artifacts, its performance under free viewing conditions has not been systematically evaluated and many published reports contain residual artifacts. Here I evaluated and optimized ICA-based correction for two tasks with unconstrained eye movements: visual search in images and sentence reading. In a first step, four parameters of the ICA pipeline were varied orthogonally: the (1) high-pass and (2) low-pass filter applied to the training data, (3) the proportion of training data containing myogenic saccadic spike potentials (SP), and (4) the threshold for eye tracker-based component rejection. In a second step, the eye-tracker was used to objectively quantify the correction quality of each ICA solution, both in terms of undercorrection (residual artifacts) and overcorrection (removal of neurogenic activity). As a benchmark, results were compared to those obtained with an alternative spatial filter, Multiple Source Eye Correction (MSEC). With commonly used settings, Infomax ICA not only left artifacts in the data, but also distorted neurogenic activity during eye movement-free intervals. However, correction results could be strongly improved by training the ICA on optimally filtered data in which SPs were massively overweighted. With optimized procedures, ICA removed virtually all artifacts, including the SP and its associated spectral broadband artifact from both viewing paradigms, with little distortion of neural activity. It also outperformed MSEC in terms of SP correction. Matlab code is provided.

The utility of micro-computed tomography for the non-destructive study of eye microstructure in snails.

Molluscan eyes exhibit an enormous range of morphological variation, ranging from tiny pigment-cup eyes in limpets, compound eyes in ark clams and pinhole eyes in Nautilus, through to concave mirror eyes in scallops and the large camera-type eyes of the more derived cephalopods. Here we assess the potential of non-destructive micro-computed tomography (µ-CT) for investigating the anatomy of molluscan eyes in three species of the family Solariellidae, a group of small, deep-sea gastropods. We compare our results directly with those from traditional histological methods applied to the same specimens, and show not only that eye microstructure can be visualised in sufficient detail for meaningful comparison even in very small animals, but also that µ-CT can provide additional insight into gross neuroanatomy without damaging rare and precious specimens. Data from µ-CT scans also show that neurological innervation of eyes is reduced in dark-adapted snails when compared with the innervation of cephalic tentacles, which are involved in mechanoreception and possibly chemoreception. Molecular tests also show that the use of µ-CT and phosphotungstic acid stain do not prevent successful downstream DNA extraction, PCR amplification or sequencing. The use of µ-CT methods is therefore highly recommended for the investigation of difficult-to-collect or unique specimens.

Simulation of visual acuity by personalizable neuro-physiological model of the human eye.

We present a model of the whole visual train to estimate an individual's visual acuity based on their eye's physical properties. Our simulation takes into account the optics of the eye, neural transmission and noise, as well as the recognition process. Personalized input data are represented by the ocular wavefront aberration and pupil diameter, both either coming from in vivo measurements of a subject or being produced by optical design software using a schematic eye. This flexibility opens the door to a broad range of potential applications, such as objective visual acuity measurements and intraocular lens design. Our algorithm contains only two adjustable neural parameters: additive noise σ, and discrimination range δρ, with their values being experimentally calibrated by fitting the results of simulations to the outcome of real acuity tests performed on healthy young subjects with normal vision (visual acuity: 0…-0.3 logMAR range). It was established that by using fixed values of σ = 0.10 and δρ = 0.0025 for each person examined, the residual of the acuity simulations averaged over the calibration group reached its minimum at 0.045 logMAR.

On the shoulders of giants: the case of the Claude Bernard Horner syndrome

The syndrome called mainly in the French world as Claude Bernard Horner was frst described by Francois Pourfour du Petit, in 1727, but more thoroughly defned by the French physiologist, Claude Bernard, in 1852, followed by several physicians who offered different interpretations, mainly Silas Weir Mitchell (1864). The clinical and pharmacological implications, with the fnal wrap-up of the syndrome, were presented by a Swiss ophthalmologist, Johann Friedrich Horner, in 1869. This is a cooperative defnition of a syndrome of the sympathetic disruption of the ocular inervation, with fnal addings mainly about pharmacological approach by Horner, but with credits to many others clinicians and physiologists. This is the case of repeated presentations of a "new sign" in neurology with few additions from one to another.

A síndrome chamada principalmente no mundo francês como Claude Bernard Horner foi descrita pela primeira vez por François Pourfour du Petit, em 1727, mas mais profundamente defnida pelo fsiologista francês, Claude Bernard, em 1852, seguido por vários médicos que ofereceram interpretações diferentes, principalmente Silas Weir Mitchell (1864). As implicações clínicas e farmacológicas, com o desfecho fnal da síndrome, foram apresentadas por um oftalmologista suíço, Johann Friedrich Horner, em 1869. Esta é uma defnição cooperativa de uma síndrome da ruptura da inervação simpática ocular, com adições fnais principalmente sobre a abordagem farmacológica por Horner, mas com créditos para muitos outros médicos e fsiologistas. É o caso de repetidas apresentações de um "novo sinal" na neurologia, com poucas adições de um para o outro.

Eyes are mirror of the brain: comparison of multiple sclerosis patients and healthy controls using OCT.

Objective: To evaluate the thickness of choroid and retinal nerve fiber layer (RNFL) in multiple sclerosis (MS) patients with and without optic neuritis using enhanced depth imaging optical coherence tomography (EDI-OCT). Methods: In this cross-sectional study, both eyes of 52 MS patients [n = 104 eyes; 62 eyes of MS patients without optic neuritis (MS-NON) and 42 eyes of MS patients with optic neuritis (MS-ON)] and only one eye of 36 healthy control subjects (n = 36 eyes) were evaluated. Complete ophthalmologic examination and EDI-OCT scanning were completed for all participants. Choroidal thickness measurements were executed at three different points. Results: Choroidal thickness measurements were similar between MS patients and healthy control subjects. However, the mean subfoveal choroidal thickness was increased significantly in MS-ON group (399.13 ± 82.91 µm) compared to MS-NON group (342.71 ± 82.46 µm; p = 0.004). Mean RNFL thickness was significantly reduced in MS patients (90.42 ± 13.31 µm) compared to healthy controls (101.18 ± 10.75 µm; p < 0.001). Moreover, temporal RNFL thickness was significantly thinner in MS-ON group (54 ± 14.50 µm) than MS-NON group (62.15 ± 15.88 µm; p = 0.01). In MS patients, temporal RNFL thickness was correlated with both Expanded Disability Status Score (r = 0.383; p < 0.001) and longer disease duration (r=-0.202; p = 0.04). Conclusion: The results of the present study suggest that RNFL thickness can be used as an important parameter while following up with MS patients. However, more studies using EDI-OCT are required with larger MS patient groups and automated method.

Chewing induced reflex seizures ("eating epilepsy") and eye closure sensitivity as a common feature in pediatric patients with SYNGAP1 mutations: Review of literature and report of 8 cases.

PURPOSE: Heterozygous SYNGAP1 gene mutations have been associated with several forms of idiopathic generalized epilepsy, autism spectrum disorders and delay of psychomotor development. We report eight patients with a SYNGAP1 mutation and chewing/eating induced reflex seizures as new phenotype and compare them to other patients with eating epilepsy and genetic mutations. METHODS: Presentation of clinical and anamnestic features and retrospective analysis of Video-EEG data of a 4 year old index patient with SYNGAP1 mutation and chewing /eating induced seizures. Clinical and anamnestic features and home videos of seven additional patients (4 female; age: 4-14 years) with SYNGAP1 mutation and eating induced reflex seizures were compared. RESULTS: All reflex seizures of the index patient showed similar focal EEG pattern with 1-5 seconds high amplitude, irregular 3/sec spike-wave complexes with initiation from left temporo-occipital, right temporo-occipital or bi- occipital / temporo-occipital regions. Eyelid myoclonia, the most common seizure type in all 8 patients, were typically initiated by eating or other simple orofacial stimuli. In the index patient eye closure preceded eating induced-eyelid myoclonia in 30/38 seizures. CONCLUSION: The main clinical features of our patient (i.e. intellectual disability, epilepsy, autistic features) are compatible with previous reports on patients with SYNGAP1 mutations. This is the first complete description of eating induced seizures in association with SYNGAP1 mutations. Whether eye closure sensitivity (ECS) represents an independent reflex epileptic trait, as seen in other patients with idiopathic "generalized" epilepsies (IGE), or eye closure is part of a complex trigger mechanism in SYNGAP1 patients' remains to be elucidated.

To roll the eyes and snap a bite - function, development and evolution of craniofacial muscles.

Craniofacial muscles, muscles that move the eyes, control facial expression and allow food uptake and speech, have long been regarded as a variation on the general body muscle scheme. However, evidence has accumulated that the function of head muscles, their developmental anatomy and the underlying regulatory cascades are distinct. This article reviews the key aspects of craniofacial muscle and muscle stem cell formation and discusses how this differs from the trunk programme of myogenesis; we show novel RNAseq data to support this notion. We also trace the origin of head muscle in the chordate ancestors of vertebrates and discuss links with smooth-type muscle in the primitive chordate pharynx. We look out as to how the special properties of head muscle precursor and stem cells, in particular their competence to contribute to the heart, could be exploited in regenerative medicine.

Vestibular-related eye movements in the rat following selective electrical stimulation of the vestibular sensors.

Rats are the most commonly used species in the neurosciences; however, little is known about the effects of selective electrical stimulation of individual vestibular sensors, on their eye movements. This limits their use to study the effects of vestibular stimulation on the brain, and their use in further exploring novel technologies such as artificial vestibular implants. We describe the effects of electrical stimulation of each vestibular sensor on vestibular-related eye movement in rats and compared the results to other species. We demonstrated that each sensor is responsible for specific bilateral eye movements. We found that the eye movements in rats differed from other species. Although the results were similar when stimulating the horizontal canal ampulla, differences appeared when stimulating the vertical canal sensors. During utricular stimulation, the ipsilateral eye moved dorsally in most cases, while the contralateral eye usually moved either caudally, or in extorsion. Saccular stimulation usually moved the ipsilateral eye dorsally or ventrally, while the contralateral eye usually moved ventrally or caudally. This study provides the first data on the application of selective electrical vestibular stimulation in the rat to the study of vestibular-related eye movements.

Length of myelin internodes of individual oligodendrocytes is controlled by microenvironment influenced by normal and input-deprived axonal activities in sensory deprived mouse models.

Oligodendrocytes myelinate neuronal axons to increase conduction velocity in the vertebrate central nervous system (CNS). Recent studies revealed that myelin formed on highly active axons is more stable compared to activity-silenced axons, and length of the myelin sheath is longer in active axons as well in the zebrafish larva. However, it is unclear whether oligodendrocytes preferentially myelinate active axons compared to sensory input-deprived axons in the adult mammalian CNS. It is also unknown if a single oligodendrocyte forms both longer myelin sheaths on active axons and shorter sheaths on input-deprived axons after long-term sensory deprivation. To address these questions, we applied simultaneous labeling of both neuronal axons and oligodendrocytes to mouse models of long-term monocular eyelid suturing and unilateral whisker removal. We found that individual oligodendrocytes evenly myelinated normal and input-deprived axons in the adult mouse CNS, and myelin sheath length on normal axons and input-deprived axons formed by a single oligodendrocyte were comparable. Importantly, the average length of the myelin sheath formed by individual oligodendrocytes did change depending on relative abundance of normal against sensory-input deprived axons, indicating an abundance of deprived axons near an oligodendrocyte impacts on myelination program by a single oligodendrocyte.

Effects of early eye removal on the morphology of a multisensory neuron in the chicken optic tectum.

The midbrain is a subcortical area involved in central functions such as integrating sensory modalities, movement initiation and bottom-up and top-down attention. In chicken, the midbrain roof is termed optic tectum (TeO) and consists of 15 layers with distinct in- and output regions. Visual input targets the superficial layers, while auditory input terminates in deeper layers. It has been shown that ablation of sensory epithelia leads to changes in the cellular patterning and structural organization of the sensory pathways. For the tectum, ablation of the eye anlagen was shown to affect retinorecipient neurons. While the gross morphology remained intact after enucleation, the shape of dendritic endings was changed presumably due to missing presynaptic input during synaptic pruning. We investigated the effect of deafferentation in a multisensory cell type, the Shepherd's crook neuron (SCN) in the TeO. SCNs have distinct dendritic branches in retinorecipient layers (superficial layers 1 to 5 and 7) and in layers where auditory input terminates. To assess whether removal of a single sensory input only affects the dendrites recipient for that input, we removed the eye anlagen and retrogradely labeled SCNs later in embryogenesis to visualize the morphology in lesioned and non-lesioned embryos. We found no changes in the gross morphology or in the basal dendrites, but an altered growth of the fine structures at the apical dendrite of SCNs in the retinorecipient layers. Our data indicate that the neuronal morphology of SCNs is mostly predefined before retinal innervation affect the fine structure.

The optic pathway: the development of an eLearning animation.

The optic pathway is responsible for sending visual information from the eyes to the brain via electrical impulses. It is essential that a sound understanding of this pathway is established in order to determine an accurate diagnosis concerning visual field defects. Although easy for trained neurologists to understand, it is an area which medical students repeatedly struggle to visualise. It is proposed that audio-visual teaching resources can improve students understanding of complex areas of importance. This article describes the development and evaluation of a short animation created for use in the undergraduate neurology curriculum at the University of Dundee School of Medicine.

Horner's syndrome following obstetric neuraxial blockade - a systematic review of the literature.

Horner's syndrome is a rarely reported complication of neuraxial blockade. In obstetric practice, the neurological signs of Horner's syndrome may cause anxiety amongst patients and healthcare staff, but more importantly may herald the onset of maternal hypotension. Medline, CINAHL, and EMBASE databases were searched to identify cases of Horner's syndrome following obstetric neuraxial blockade. Anaesthetic technique, clinical features, anaesthetic management of the Horner's syndrome and time to resolution were assessed. Seventy-eight case reports of Horner's syndrome following obstetric neuraxial blockade were identified. Nine cases also had trigeminal nerve palsy and one case had hypoglossal nerve palsy. Amongst the 78 cases, 74% developed Horner's syndrome within one hour of a local anaesthetic bolus. The median time for resolution of Horner's syndrome was two hours, though one case was permanent. One case of Horner's syndrome was found to be due to an internal carotid artery dissection. Some cases of Horner's syndrome resolved spontaneously despite ongoing administration of epidural local anaesthetic. Hypotension was reported in 13%. Horner's syndrome is usually a benign phenomenon, the consequence of high cephalad spread of local anaesthetic, that resolves spontaneously within a few hours. Patients with a persistent Horner's syndrome, or one associated with atypical features such as neck pain, should undergo a diagnostic workup including magnetic resonance angiography of the neck. The dermatomal level of neuraxial blockade, maternal and fetal well-being should be taken into account when making decisions regarding neuraxial blockade. The presence of Horner's syndrome alone should not lead to discontinuation of neuraxial blockade.

A novel map of the mouse eye for orienting retinal topography in anatomical space.

Functionally distinct retinal ganglion cells have density and size gradients across the mouse retina, and some degenerative eye diseases follow topographic-specific gradients of cell death. Hence, the anatomical orientation of the retina with respect to the orbit and head is important for understanding the functional anatomy of the retina in both health and disease. However, different research groups use different anatomical landmarks to determine retinal orientation (dorsal, ventral, temporal, nasal poles). Variations in the accuracy and reliability in marking these landmarks during dissection may lead to discrepancies in the identification and reporting of retinal topography. The goal of this study was to compare the accuracy and reliability of the canthus, rectus muscle, and choroid fissure landmarks in reporting retinal orientation. The retinal relieving cut angle made from each landmark during dissection was calculated based on its relationship to the opsin transition zone (OTZ), determined via a custom MATLAB script that aligns retinas from immunostained s-opsin. The choroid fissure and rectus muscle landmarks were the most accurate and reliable, while burn marks using the canthus as a reference were the least. These values were used to build an anatomical map that plots various ocular landmarks in relationship to one another, to the horizontal semicircular canals, to lambda-bregma, and to the earth's horizon. Surprisingly, during normal locomotion, the mouse's opsin gradient and the horizontal semicircular canals make equivalent 6° angles aligning the OTZ near the earth's horizon, a feature which may enhance the mouse's ability to visually navigate through its environment.

Perineurial-like Cells and EMA Expression in the Suprachoroidal Region of the Human Eye.

The suprachoroidal region of the eye comprises vascular channels, melanocytes, and thin fibroblasts with elongated cytoplasm that are positioned directly adjacent to the densely collagenous sclera. Morphological similarities between these suprachoroidal fibroblasts and arachnoid cells and perineurial cells have been recognized, but whether these fibroblasts have a perineurial cell-like immunophenotype is not known. To further examine the relationship of these three cell types, we investigated the comparative expression of epithelial membrane antigen (EMA), the tight junction protein claudin-1, glucose transporter-1 (Glut-1), and CD34 in suprachoroidal fibroblasts, arachnoid of the optic nerve sheath, and perineurium of ciliary nerves in eight human eye specimens. Granular, diffuse, and cytoplasmic EMA expression was seen in suprachoroidal fibroblasts, but this was not contiguous with the similar pattern of EMA expression in adjacent perineurium and arachnoid. CD34 expression in suprachoroidal fibroblasts was also seen, similar to arachnoid and perineurium. Claudin-1 and Glut-1 were not consistently expressed in suprachoroidal fibroblasts, distinguishing them from perineurial cells in particular and suggesting that these fibroblasts do not arise directly from adjacent arachnoid or perineurium. Nonetheless, the overlapping morphology and protein expression suggest phenotypic similarities in these cells that protect and support adjacent retina, optic nerve, and peripheral nerve.

Eye-specific segregation and differential fasciculation of developing retinal ganglion cell axons in the mouse visual pathway.

Prior to forming and refining synaptic connections, axons of projection neurons navigate long distances to their targets. While much is known about guidance cues for axon navigation through intermediate choice points, whether and how axons are organized within tracts is less clear. Here we analyze the organization of retinal ganglion cell (RGC) axons in the developing mouse retinogeniculate pathway. RGC axons are organized by both eye-specificity and topography in the optic nerve and tract: ipsilateral RGC axons are segregated from contralateral axons and are offset laterally in the tract relative to contralateral axon topographic position. To identify potential cell-autonomous factors contributing to the segregation of ipsilateral and contralateral RGC axons in the visual pathway, we assessed their fasciculation behavior in a retinal explant assay. Ipsilateral RGC neurites self-fasciculate more than contralateral neurites in vitro and maintain this difference in the presence of extrinsic chiasm cues. To further probe the role of axon self-association in circuit formation in vivo, we examined RGC axon organization and fasciculation in an EphB1-/- mutant, in which a subset of ipsilateral RGC axons aberrantly crosses the midline but targets the ipsilateral zone in the dorsal lateral geniculate nucleus on the opposite side. Aberrantly crossing axons retain their association with ipsilateral axons in the contralateral tract, indicating that cohort-specific axon affinity is maintained independently of guidance signals present at the midline. Our results provide a comprehensive assessment of RGC axon organization in the retinogeniculate pathway and suggest that axon self-association contributes to pre-target axon organization.

Horner Syndrome in 2 Pigs (Sus scrofa) after Vascular Grafting of the Carotid Artery and Jugular Vein.

The term Horner syndrome refers to the clinical presentation of oculosympathoparesis, comprising miosis, ptosis, and facial anhydrosis. To date, there are 2 reports of postoperative Horner syndrome in pigs. In this species the cervical sympathetic chain and cranial cervical sympathetic ganglion are consistently within the carotid artery sheath. This case study describes the sudden onset of Horner syndrome in 2 pigs, from a study cohort of 8, after the placement of a vascular graft between the carotid artery and external jugular vein. Anesthesia and surgery was uneventful in all the pigs in the study, but 2 pigs demonstrated clinical signs including ptosis, enophthalmos and prolapse of the nictitating membrane immediately after recovery from anesthesia. Horner syndrome was diagnosed in light of the clinical signs. These clinical signs persisted throughout the 2-mo study period and did not appear to improve or deteriorate in that time. Gross examination of the surgery site at the end of the study did not reveal an obvious lesion in the carotid artery sheath. The risk of Horner syndrome after surgery involving the carotid artery in pigs had not been reported prior to this study. Without specific measures to protect the cervical sympathetic ganglion during surgery, the incidence of postoperative Horner syndrome was 25% in our population of pigs. Although the welfare implications of this syndrome are minimal, concerted effort to avoid intraoperative damage to the cervical ganglion is essential for future work.

Elevated expression of neuropeptide signaling genes in the eyestalk ganglia and Y-organ of Gecarcinus lateralis individuals that are refractory to molt induction.

Molting is induced in decapod crustaceans via multiple leg autotomy (MLA) or eyestalk ablation (ESA). MLA removes five or more walking legs, which are regenerated and become functional appendages at ecdysis. ESA eliminates the primary source of molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), which suppress the production of molting hormones (ecdysteroids) from the molting gland or Y-organ (YO). Both MLA and ESA are effective methods for molt induction in Gecarcinus lateralis. However, some G. lateralis individuals are refractory to MLA, as they fail to complete ecdysis by 12weeks post-MLA; these animals are in the "blocked" condition. Quantitative polymerase chain reaction was used to quantify mRNA levels of neuropeptide and mechanistic target of rapamycin (mTOR) signaling genes in YO, eyestalk ganglia (ESG), thoracic ganglion (TG), and brain of intact and blocked animals. Six of the seven neuropeptide signaling genes, three of four mTOR signaling genes, and Gl-elongation factor 2 (EF2) mRNA levels were significantly higher in the ESG of blocked animals. Gl-MIH and Gl-CHH mRNA levels were higher in the TG and brain of blocked animals and levels increased in both control and blocked animals in response to ESA. By contrast, mRNA levels of Gl-EF2 and five of the 10 MIH signaling pathway genes in the YO were two to four orders of magnitude higher in blocked animals compared to controls. These data suggest that increased MIH and CHH synthesis in the ESG contributes to the prevention of molt induction by MLA in blocked animals. The up-regulation of MIH signaling genes in the YO of blocked animals suggests that the YO is more sensitive to MIH produced in the ESG, as well as MIH produced in brain and TG of ESA animals. Both the up-regulation of MIH signaling genes in the YO and of Gl-MIH and Gl-CHH in the ESG, TG, and brain appear to contribute to some G. lateralis individuals being refractory to MLA and ESA.