Permanent transduction of retinal ganglion cells by rAAV2-retro.

Adeno-associated virus (AAV) is widely used as a vector for delivery of gene therapy. Long term therapeutic benefit depends on perpetual expression of the wild-type gene after transduction of host cells by AAV. To address this issue in a mass population of identified single cells, 4 rats received an injection of a 1:1 mixture of rAAV2-retro-hSyn-EGFP and rAAV2-retro-hSyn-mCherry into each superior colliculus. After the virus was transported retrogradely to both retinas, serial fundus imaging was performed at days 14, 45, 211, and 375 to visualize individual fluorescent ganglion cells. The location of each cell was plotted to compare labeling at each time point. In 12/16 comparisons, 97% or more of the cells identified in the initial baseline fundus image were still labeled at day 375. In 4 cases the percentage was lower, but in these cases the apparent reduction in the number of labeled cells at day 375 was attributable to the lower quality of follow-up fundus images, rather than true loss of transgene expression. These data indicate that retinal ganglion cells transduced by rAAV2-retro are transduced permanently.

Revised Diagnosis From Histiocytic Neoplasm to Optic Chiasm Glioblastoma After Genetic Analysis.

ABSTRACT: A 46-year-old man presented with left eye blurring. Automated visual field testing showed an incongruous right hemianopia, with sparing of the lower temporal quadrant in the right eye. MRI revealed foci of gadolinium enhancement in the optic chiasm and optic tracts. Serologic testing (including myelin oligodendrocyte glycoprotein and neuromyelitis optica antibodies) and cerebrospinal fluid analysis were negative. Whole-body PET/CT scan found no malignancy. Biopsy of the optic chiasm revealed a moderately cellular neoplasm composed of atypical, discohesive cells with enlarged nuclei, prominent eosinophilic nucleoli, and abundant vacuolated cytoplasm. Immunohistochemical stains for CD68 and S100 were positive, whereas those for GFAP, OLIG2, SOX10, and multiple others were negative, supporting a diagnosis of histiocytic neoplasm. Five weeks later, results became available from next-generation sequencing targeting the coding regions of hundreds of malignancy-associated genes and select introns. Alterations associated with histiocytic neoplasms (i.e. BRAF and MAP2K1 mutations) were absent. However, there was a nonsense mutation in the PTEN gene, a hotspot mutation in the TERT gene promotor, and focal amplifications of the CDK4 and MDM2 genes. Additionally, there was chromosome 6q loss, 7 gain, and 10q loss. Based on these findings, the diagnosis was revised to glioblastoma, IDH-wildtype, CNS WHO grade 4. The patient began treatment with temozolomide while continuing radiation therapy. This case illustrates how next-generation sequencing can at times provide more accurate diagnostic information than standard tissue histopathology.

Interocular Suppression in Primary Visual Cortex in Strabismus.

People with strabismus acquired during childhood do not experience diplopia (double vision). To investigate how perception of the duplicate image is suppressed, we raised two male monkeys with alternating exotropia by disinserting the medial rectus muscle in each eye at age four weeks. Once the animals were mature, they were brought to the laboratory and trained to fixate a small spot while recordings were made in primary visual cortex (V1). Drifting gratings were presented to the receptive fields of 500 single neurons for eight interleaved conditions: (1) right eye monocular; (2) left eye monocular; (3) right eye's field, right eye fixating; (4) right eye's field, left eye fixating; (5) left eye's field, right eye fixating; (6) left eye's field, left eye fixating; (7) both eyes' fields, right eye fixating; (8) both eyes' fields, left eye fixating. As expected, ocular dominance histograms showed a monocular bias compared with normal animals, but many cells could still be driven via both eyes. Overall, neuronal responses were not affected by switches in ocular fixation. Individual neurons exhibited binocular interactions, but mean population indices indicated no net interocular suppression or facilitation. Even neurons located in cortex with reduced cytochrome oxidase (CO) activity, representing portions of the nasal visual field where perception is suppressed during binocular viewing, showed no net inhibition. These data indicate that V1 neurons do not appear to reflect strabismic suppression and therefore the elimination of diplopia is likely to be mediated at a higher cortical level.SIGNIFICANCE STATEMENT In patients with strabismus, images fall on non-corresponding points in the two retinas. Only one image is perceived, because signals emanating from the other eye that convey the duplicate image are suppressed. The benefit is that diplopia is prevented, but the penalty is that the visual feedback required to adjust eye muscle tone to realign the globes is eliminated. Here, we report the first electrophysiological recordings from the primary visual cortex (V1) in awake monkeys raised with strabismus. The experiments were designed to reveal how perception of double images is avoided.

Fundus imaging of retinal ganglion cells transduced by retrograde transport of rAAV2-retro.

Access of adeno-associated virus (AAV) to ganglion cells following intravitreal injection for gene therapy is impeded by the internal limiting membrane of the retina. As an alternative, one could transduce ganglion cells via retrograde transport after virus injection into a retinal target nucleus. It is unknown if recombinant AAV2-retro (rAAV2-retro), a variant of AAV2 developed specifically for retrograde transport, is capable of transducing retinal ganglion cells. To address this issue, equal volumes of rAAV2-retro-hSyn-EGFP and rAAV2-retro-hSyn-mCherry were mixed in a micropipette and injected into the rat superior colliculus. The time-course of viral transduction was tracked by performing serial in vivo fundus imaging. Cells that were labeled by the fluorophores within the first week remained consistent in distribution and relative signal strength on follow-up imaging. Most transduced cells were double-labeled, but some were labeled by only EGFP or mCherry. Fundus images were later aligned with retinal wholemounts. Ganglion cells in the wholemounts matched precisely the cells imaged by fundus photography. As seen in the fundus images, ganglion cells in wholemounts were sometimes labeled by only EGFP or mCherry. Overall, there was detectable label in 32-41% of ganglion cells. Analysis of the number of cells labeled by 0, 1, or 2 fluorophores, based on Poisson statistics, yielded an average of 0.66 virions transducing each ganglion cell. Although this represents a low number relative to the quantity of virus injected into the superior colliculus, the ganglion cells showed sustained and robust fluorescent labeling. In the primate, injection of rAAV2-retro into the lateral geniculate nucleus might provide a viable approach for the transduction of ganglion cells, bypassing the obstacles that have prevented effective gene delivery via intravitreal injection.

Retinal Input to the Primate Lateral Geniculate Nucleus Revealed by Injection of a Different Label Into Each Eye.

ABSTRACT: The primate lateral geniculate nucleus has long been a favorite structure among anatomists because of its striking lamination. It has been shown that each lamina receives input from a different eye using various single label techniques but never by double labeling. Here, we illustrate the organization of retinal inputs to the lateral geniculate nucleus by injection of cholera toxin-B conjugated to Alexa Fluor-488 into the right eye and cholera toxin-B conjugated to Alexa Fluor-594 into the left eye.

Interocular suppression in primary visual cortex in strabismus: impact of staggering the presentation of stimuli to the eyes.

Diplopia (double vision) in strabismus is prevented by suppression of the image emanating from one eye. In a recent study conducted in two macaques raised with exotropia (an outward ocular deviation) but having normal acuity in each eye, simultaneous display of stimuli to each eye did not induce suppression in V1 neurons. Puzzled by this negative result, we have modified our protocol to display stimuli in a staggered sequence, rather than simultaneously. Additional recordings were made in the same two macaques, following two paradigms. In trial type 1, the receptive field in one eye was stimulated with a sine-wave grating while the other eye was occluded. After 5 s, the occluder was removed and the neuron was stimulated for another 5 s. The effect of uncovering the eye, which potentially exposed the animal to diplopia, was quantified by the peripheral retinal interaction index (PRII). In trial type 2, the receptive field in the fixating eye was stimulated with a grating during binocular viewing. After 5 s, a second grating appeared in the receptive field of the nonfixating eye. The impact of the second grating, which had the potential to generate visual confusion, was quantified by the receptive field interaction index (RFII). For 82 units, the mean PRII was 0.48 ± 0.05 (0.50 = no suppression) and the mean RFII was 0.46 ± 0.08 (0.50 = no suppression). These values suggest mild suppression, but the modest decline in spike rate registered during the second epoch of visual stimulation might have been due to neuronal adaptation, rather than interocular suppression. In a few instances neurons showed unequivocal suppression, but overall, these recordings did not support the contention that staggered stimulus presentation is more effective than simultaneous stimulus presentation at evoking interocular suppression in V1 neurons.NEW & NOTEWORTHY In strabismus, double vision is prevented by interocular suppression. It has been reported that inhibition of neuronal firing in the primary visual cortex occurs only when stimuli are presented sequentially, rather than simultaneously. However, these recordings in alert macaques raised with exotropia showed, with rare exceptions, little evidence to support the concept that staggered stimulus presentation is more effective at inducing interocular suppression of V1 neurons.

Congenital Visual Field Loss from a Schizencephalic Cleft Damaging Meyer's Loop.

A healthy, asymptomatic woman was referred after incidental discovery of a right superior incongruous hemianopia. Magnetic resonance imaging disclosed a schizencephalic cleft passing through Meyer's loop of the left optic radiation. The lesion may have resulted from a focal vascular accident or disruption of cortical neurogenesis during gestation.

Wilbrand's Knee: To Be or Not to Be a Knee?

Wilbrand's knee of the optic chiasm refers to crossing fibers from one optic nerve that stray for a short distance into the opposite optic nerve before joining the optic tract. This loop of aberrant axons, although small, has generated much controversy. In a previous study, labeling of the optic pathway in normal monkeys with a radioactive tracer revealed no Wilbrand's knee. Monocular enucleation induced a typical knee to form. These findings suggested that Wilbrand's knee is absent normally, but appears after atrophy of one optic nerve. This conclusion has been challenged by images showing Wilbrand's knee in the normal human chiasm using anisotropic light scattering. It has also been resisted by some clinicians who believe that Wilbrand's knee is necessary to explain the anterior chiasmal syndrome. Early in his distinguished career, William F. Hoyt examined the fiber organization of the monkey optic nerve and chiasm. He found no evidence for Wilbrand's knee and rejected its importance for the topical diagnosis of chiasmal lesions. His conclusion is supported by new data showing that anisotropic light scattering is not a reliable method for tracing axons. Hence, that method has given a misleading impression that Wilbrand's knee exists in normal subjects. Although Wilbrand's knee has fascinated neuro-ophthalmologists for more than a century, it is an inconsequential structure that develops only after a longstanding monocular optic neuropathy.

Normal Topography and Binocularity of the Superior Colliculus in Strabismus.

In subjects with alternating strabismus, either eye can be used to saccade to visual targets. The brain must calculate the correct vector for each saccade, which will depend on the eye chosen to make it. The superior colliculus, a major midbrain center for saccade generation, was examined to determine whether the maps serving each eye were shifted to compensate for strabismus. Alternating exotropia was induced in two male macaques at age 1 month by sectioning the tendons of the medial recti. Once the animals grew to maturity, they were trained to fixate targets with either eye. Receptive fields were mapped in the superior colliculus using a sparse noise stimulus while the monkeys alternated fixation. For some neurons, sparse noise was presented dichoptically to probe for anomalous retinal correspondence. After recordings, microstimulation was applied to compare sensory and motor maps. The data showed that receptive fields were offset in position by the ocular deviation, but otherwise remained aligned. In one animal, the left eye's coordinates were rotated ∼20° clockwise with respect to those of the right eye. This was explained by a corresponding cyclorotation of the ocular fundi, which produced an A-pattern deviation. Microstimulation drove the eyes accurately to the site of receptive fields, as in normal animals. Single-cell recordings uncovered no evidence for anomalous retinal correspondence. Despite strabismus, neurons remained responsive to stimulation of either eye. Misalignment of the eyes early in life does not alter the organization of topographic maps or disrupt binocular convergence in the superior colliculus.SIGNIFICANCE STATEMENT Patients with strabismus are able to make rapid eye movements, known as saccades, toward visual targets almost as gracefully as subjects with normal binocular alignment. They can even exercise the option of using the right eye or the left eye. It is unknown how the brain measures the degree of ocular misalignment and uses it to compute the appropriate saccade for either eye. The obvious place to investigate is the superior colliculus, a midbrain oculomotor center responsible for the generation of saccades. Here, we report the first experiments in the superior colliculus of awake primates with strabismus using a combination of single-cell recordings and microstimulation to explore the organization of its topographic maps.

Recurrent Superior Oblique Myokymia Treated by Distal Tendon Extirpation.

BACKGROUND: We describe successful surgical treatment of superior oblique myokymia, which had recurred after superior oblique tenectomy. METHODS: Single case report. RESULTS: The distal stump of the superior oblique tendon was extirpated by stripping it from the globe. The ipsilateral superior rectus muscle also was recessed, to correct a hypertropia that had resulted from the original superior oblique tenectomy. CONCLUSIONS: Complete removal of the distal superior oblique muscle tendon provided definitive relief of superior oblique myokymia. Superior rectus muscle recession, combined with previous inferior oblique myectomy, compensated effectively for loss of superior oblique function.

Patterns of Cortical Visual Field Defects From Embolic Stroke Explained by the Anastomotic Organization of Vascular Microlobules.

The cerebral cortex is supplied by vascular microlobules, each comprised of a half dozen penetrating arterioles that surround a central draining venule. The surface arterioles that feed the penetrating arterioles are interconnected via an extensively anastomotic plexus. Embolic occlusion of a small surface arteriole rarely produces a local infarct, because collateral blood flow is available through the vascular reticulum. Collateral flow also protects against infarct after occlusion of a single penetrating arteriole. Cortical infarction requires blockage of a major arterial trunk, with arrest of blood flow to a relatively large vascular territory. For striate cortex, the major vessels compromised by emboli are the inferior calcarine and superior calcarine arteries, as well as the distal branches of the middle cerebral artery. Their vascular territories have a fairly consistent relationship with the retinotopic map. Consequently, occlusion by emboli results in stereotypical visual field defects. The organization of the arterial supply to the occipital lobe provides an anatomical explanation for a phenomenon that has long puzzled neuro-ophthalmologists, namely, that of the myriad potential patterns of cortical visual field loss, only a few are encountered commonly from embolic cortical stroke.

Capturing the Moment of Fusion Loss in Intermittent Exotropia.

PURPOSE: To characterize eye movements made by patients with intermittent exotropia when fusion loss occurs spontaneously and to compare them with those induced by covering 1 eye and with strategies used to recover fusion. DESIGN: Prospective study of a patient cohort referred to our laboratory. PARTICIPANTS: Thirteen patients with typical findings of intermittent exotropia who experienced frequent spontaneous loss of fusion. METHODS: The position of each eye was recorded with a video eye tracker under infrared illumination while fixating on a small central near target. MAIN OUTCOME MEASURES: Eye position and peak velocity measured during spontaneous loss of fusion, shutter-induced loss of fusion, and recovery of fusion. RESULTS: In 10 of 13 subjects, the eye movement made after spontaneous loss of fusion was indistinguishable from that induced by covering 1 eye. It reached 90% of full amplitude in a mean of 1.75 seconds. Peak velocity of the deviating eye's movement was highly correlated for spontaneous and shutter-induced events. Peak velocity was also proportional to exotropia amplitude. Recovery of fusion was more rapid than loss of fusion, and often was accompanied by interjection of a disconjugate saccade. CONCLUSIONS: Loss of fusion in intermittent exotropia is not influenced by visual feedback. Excessive divergence tone may be responsible, but breakdown of alignment occurs via a unique, pathological type of eye movement that differs from a normal, physiological divergence eye movement.

Adaptation, perceptual learning, and plasticity of brain functions.

The capacity for functional restitution after brain damage is quite different in the sensory and motor systems. This series of presentations highlights the potential for adaptation, plasticity, and perceptual learning from an interdisciplinary perspective. The chances for restitution in the primary visual cortex are limited. Some patterns of visual field loss and recovery after stroke are common, whereas others are impossible, which can be explained by the arrangement and plasticity of the cortical map. On the other hand, compensatory mechanisms are effective, can occur spontaneously, and can be enhanced by training. In contrast to the human visual system, the motor system is highly flexible. This is based on special relationships between perception and action and between cognition and action. In addition, the healthy adult brain can learn new functions, e.g. increasing resolution above the retinal one. The significance of these studies for rehabilitation after brain damage will be discussed.

Bilateral Optic Disc Pits With Posterior Pituitary Ectopia.

Posterior pituitary ectopia has been reported previously in association with optic nerve hypoplasia, as a variant of septo-optic dysplasia. We describe a 14-year-old boy with posterior pituitary ectopia and bilateral optic disc pits. He had hypopituitarism and a reduction in visual acuity to 20/40 in each eye, owing to loss of foveal ganglion cells. Optic pits and posterior pituitary ectopia may have occurred together in the same subject by chance, but the rarity of both conditions suggests a possible association.

Normal correspondence of tectal maps for saccadic eye movements in strabismus.

The superior colliculus is a major brain stem structure for the production of saccadic eye movements. Electrical stimulation at any given point in the motor map generates saccades of defined amplitude and direction. It is unknown how this saccade map is affected by strabismus. Three macaques were raised with exotropia, an outwards ocular deviation, by detaching the medial rectus tendon in each eye at age 1 mo. The animals were able to make saccades to targets with either eye and appeared to alternate fixation freely. To probe the organization of the superior colliculus, microstimulation was applied at multiple sites, with the animals either free-viewing or fixating a target. On average, microstimulation drove nearly conjugate saccades, similar in both amplitude and direction but separated by the ocular deviation. Two monkeys showed a pattern deviation, characterized by a systematic change in the relative position of the two eyes with certain changes in gaze angle. These animals' saccades were slightly different for the right eye and left eye in their amplitude or direction. The differences were consistent with the animals' underlying pattern deviation, measured during static fixation and smooth pursuit. The tectal map for saccade generation appears to be normal in strabismus, but saccades may be affected by changes in the strabismic deviation that occur with different gaze angles.

Cortical Representation of a Myopic Peripapillary Crescent.

PURPOSE: To determine how formation of an acquired myopic crescent adjacent to the optic disc affects metabolic activity in the primary visual cortex. DESIGN: Laboratory animal study. PARTICIPANTS: Three macaque monkeys. METHODS: The blind spot region in the primary visual cortex was labeled by cytochrome oxidase (CO) histochemistry analysis or [(3)H]proline autoradiography. MAIN OUTCOME MEASURES: Visualization of the representation of the blind spot and myopic peripapillary crescent in the visual cortex. RESULTS: In high myopia, a region resembling the myopic peripapillary crescent was visible in cortical sections processed for CO. In this region, metabolic activity was reduced in ocular dominance columns that normally would be driven by input from retina corresponding to the myopic peripapillary crescent. CONCLUSIONS: The formation of a myopic crescent is accompanied by loss of metabolic activity in the cortex supplied by the affected retina. This observation confirms that retinal tissue is damaged by the development of a myopic crescent, rather than simply translocated in a temporal direction. The cortical defect matches the myopic peripapillary crescent in size and shape, indicating that fill-in of the retinotopic map by healthy, surrounding retina does not occur.

Vascular Supply of the Cerebral Cortex is Specialized for Cell Layers but Not Columns.

The vascular supply to layers and columns was compared in macaque primary visual cortex (V1) by labeling red blood cells via their endogenous peroxidase activity. Alternate sections were processed for cytochrome oxidase to reveal "patches" or "blobs," which anchor the interdigitated column systems of striate cortex. More densely populated cell layers received the most profuse blood supply. In the superficial layers the blood supply was organized into microvascular lobules, consisting of a central venule surrounded by arterioles. Each vessel was identified as an arteriole or venule by matching it with the penetration site where it entered the cortex from a parent arteriole or venule in the pial circulation. Although microvascular lobules and cytochrome oxidase patches had a similar periodicity, they bore no mutual relationship. The size and density of penetrating arterioles and venules did not differ between patches and interpatches. The red blood cell labeling in patches and interpatches was equal. Moreover, patches and interpatches were supplied by an anastomotic pial arteriole system, with no segregation of blood supply to the two compartments. Often a focal constriction was present at the origin of pial arterial branches, indicating that local control of cortical perfusion may be accomplished by vascular sphincters.

Diagnosis of Tensilon-Negative Ocular Myasthenia Gravis By Daily Selfie.

The initial symptoms of myasthenia gravis are usually ptosis and diplopia. The diagnosis is often confirmed by testing for anti-acetylcholine receptor antibodies or by observing the effects of intravenous edrophonium (Tensilon) injection. However, these standard tests may be negative in patients with isolated ocular findings. We present the case of an 83-year-old woman with negative serologic and Tensilon testing. She was asked to photograph herself daily. The resulting sequence of daily selfies captured striking fluctuations in her ocular alignment and ptosis. Daily selfies may be a useful strategy for confirming the clinical diagnosis of ocular myasthenia gravis.

Eye choice for acquisition of targets in alternating strabismus.

In strabismus, potentially either eye can inform the brain about the location of a target so that an accurate saccade can be made. Sixteen human subjects with alternating exotropia were tested dichoptically while viewing stimuli on a tangent screen. Each trial began with a fixation cross visible to only one eye. After the subject fixated the cross, a peripheral target visible to only one eye flashed briefly. The subject's task was to look at it. As a rule, the eye to which the target was presented was the eye that acquired the target. However, when stimuli were presented in the far nasal visual field, subjects occasionally performed a "crossover" saccade by placing the other eye on the target. This strategy avoided the need to make a large adducting saccade. In such cases, information about target location was obtained by one eye and used to program a saccade for the other eye, with a corresponding latency increase. In 10/16 subjects, targets were presented on some trials to both eyes. Binocular sensory maps were also compiled to delineate the portions of the visual scene perceived with each eye. These maps were compared with subjects' pattern of eye choice for target acquisition. There was a correspondence between suppression scotoma maps and the eye used to acquire peripheral targets. In other words, targets were fixated by the eye used to perceive them. These studies reveal how patients with alternating strabismus, despite eye misalignment, manage to localize and capture visual targets in their environment.

Contrasting effects of strabismic amblyopia on metabolic activity in superficial and deep layers of striate cortex.

To probe the mechanism of visual suppression, we have raised macaques with strabismus by disinserting the medial rectus muscle in each eye at 1 mo of age. Typically, this operation produces a comitant, alternating exotropia with normal acuity in each eye. Here we describe an unusual occurrence: the development of severe amblyopia in one eye of a monkey after induction of exotropia. Shortly after surgery, the animal demonstrated a strong fixation preference for the left eye, with apparent suppression of the right eye. Later, behavioral testing showed inability to track or to saccade to targets with the right eye. With the left eye occluded, the animal demonstrated no visually guided behavior. Optokinetic nystagmus was absent in the right eye. Metabolic activity in striate cortex was assessed by processing the tissue for cytochrome oxidase (CO). Amblyopia caused loss of CO in one eye's rows of patches, presumably those serving the blind eye. Layers 4A and 4B showed columns of reduced CO, in register with pale rows of patches in layer 2/3. Layers 4C, 5, and 6 also showed columns of CO activity, but remarkably, comparison with more superficial layers showed a reversal in contrast. In other words, pale CO staining in layers 2/3, 4A, and 4B was aligned with dark CO staining in layers 4C, 5, and 6. No experimental intervention or deprivation paradigm has been reported previously to produce opposite effects on metabolic activity in layers 2/3, 4A, and 4B vs. layers 4C, 5, and 6 within a given eye's columns.