Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates.

Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber's congenital amaurosis and choroideremia. One general challenge for AAV is that preexisting immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizing antibodies (NABs) in AAV transduction of tissues considered to be immune privileged, such as the eye, is unclear in large animals. Intravitreal AAV administration allows for broad retinal delivery, but is more susceptible to interactions with the immune system than subretinal administration. To assess the effects of systemic anti-AAV antibody levels on intravitreal gene delivery, we quantified the anti-AAV antibodies present in sera from non-human primates before and after intravitreal injections with various AAV capsids. Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene or dosage of virus injected. For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered. In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were also cross-reactive against other AAV serotypes. Furthermore, NAB levels in serum correlated with those in vitreal fluid, demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system and that serum measurements are predictive of vitreous fluid NAB titers. Moreover, the presence of preexisting NAB titers in the serum of monkeys correlated strongly (R=0.76) with weak, decaying or no transgene expression following intravitreal administration of AAV. Investigating anti-AAV antibody development will aid in understanding the interactions between gene therapy vectors and the immune system during ocular administration and can form a basis for future clinical studies applying intravitreal gene delivery.

Imaging retinal mosaics in the living eye.

Adaptive optics imaging of cone photoreceptors has provided unique insight into the structure and function of the human visual system and has become an important tool for both basic scientists and clinicians. Recent advances in adaptive optics retinal imaging instrumentation and methodology have allowed us to expand beyond cone imaging. Multi-wavelength and fluorescence imaging methods with adaptive optics have allowed multiple retinal cell types to be imaged simultaneously. These new methods have recently revealed rod photoreceptors, retinal pigment epithelium (RPE) cells, and the smallest retinal blood vessels. Fluorescence imaging coupled with adaptive optics has been used to examine ganglion cells in living primates. Two-photon imaging combined with adaptive optics can evaluate photoreceptor function non-invasively in the living primate retina.

Perceptual deficits after lesions of inferotemporal cortex in macaques.

This study used a novel approach to examine a much studied question, the nature of visual deficits caused by lesions of the inferotemporal cortex (IT). Unlike many previous studies of IT lesions, we de-emphasized early, non-specific disruptions of testing caused by the lesions, and instead concentrated on permanent changes in thresholds. This approach produced unexpected results that suggest a re-evaluation of the traditional view of the role of the IT cortex in shape perception and such related visual abilities as perceptual invariances, visual grouping, the visibility of illusory contours and the performance of oddity discriminations. In addition, the measurement of stable, post-lesion hue discrimination thresholds gave us a different perspective on the severity of color vision deficits which result from lesions of the IT cortex. We found that shape distortion thresholds were not permanently elevated by IT lesions and, indeed, showed no greater transitory disruption than did other visual abilities. This result is inconsistent with the common view that IT is critical to shape discriminations. Two other visual abilities that would be expected to be disrupted by IT lesions - the visual grouping of misoriented line segments and shape invariances (failure of irrelevant stimulus changes to disrupt shape distortion thresholds) - were not affected by IT lesions. However, shape discriminations based on illusory contours and some oddity discriminations were severely and permanently affected. Our results also showed that IT lesions caused permanent, moderate to large impairments of color vision, but not color blindness. Bilateral damage to area TEO caused no disruption of performance on any of the abovediscriminations. Our results suggest that the IT cortex in macaques may be critical to the visibility of illusory contours and the performance of some oddity discriminations, that it plays some role in color perception, but that it is not essential for shape, grouping discriminations or perceptual shape invariances.

Cortical area V4 is critical for certain texture discriminations, but this effect is not dependent on attention.

This study examined the question of which features of a complex grouping discrimination make it vulnerable to permanent elimination by V4 lesions. We first verified that the line element grouping discrimination, which we previously reported to be devastated by V4 lesions, was similarly affected in the monkeys of this study. The permanence of the deficit was established by mapping its visual field distribution and then testing this discrimination for an extended period at a locus on the border of the deficit. Also, a staircase procedure was used to provide the monkey with within session instruction in the grouping discrimination, but this did not improve V4 lesion performance. Grouping was then compared with several discriminations that shared some features with it, but which were found not to be permanently eliminated by V4 lesions. This comparison suggested that grouping (rather than segmentation or response to a single element) was one feature that made the discrimination vulnerable, a second was the similarity in shape of the texture elements to be grouped. Finally, we tested visual crowding, a property of peripheral vision that is thought to reflect neuronal interactions early in visual cortex, possibly in area V1, and found no effect of V4 lesions. A control experiment with human observers tested whether the elimination of grouping by V4 lesions might be due to an alteration of attention, but found no evidence to support this hypothesis. These results show that severe disruption of texture discriminations by V4 lesions depends on both the nature of the discrimination and the type of texture elements involved, but does not necessarily involve the disruption of attention.

Deficits in complex visual perception following unilateral temporal lobectomy.

Although human temporal cortex is known to be important for short- and long-term memory, its role in visual perception is not well understood. In this study, we compared the performance of three patients with unilateral temporal lobectomies to that of normal controls on both "simple" and "complex" visual discriminations that did not involve explicit memory components. Two types of complex tasks were tested that involved discriminations secondary to texture segmentation. These were contrasted with simple discriminations using luminance-defined stimuli. Patients showed impaired thresholds only on tasks involving texture segmentation, performing as well as controls when the targets were defined by luminance rather than texture. The minimum stimulus presentation times for threshold performance were also measured for all tasks and found to be elevated in temporal lobectomy patients relative to controls. Although the magnitude of the deficits observed was substantial, loss was equivalent in ipsi- and contra-lesional regions of the visual field. Additional control experiments showed that the patients' perceptual deficits were not due, even in part, to disturbances of basic visual capacities such as acuity and contrast sensitivity. Our results indicate that temporal lobe damage disrupts complex, but not simple, visual discriminations throughout the visual field.

V4 lesions in macaques affect both single- and multiple-viewpoint shape discriminations.

The role of cortical area V4 in complex shape discriminations was studied by testing the effects of V4 lesions in macaques on the ability to visually discriminate between images of three-dimensional (3D) objects from different viewpoints. Stimuli were presented in pairs in the lower left or lower right visual field quadrants about 4 deg from the fovea, and the monkeys judged on each trial whether the two views were of the same or of different objects. Object similarity was varied to determine a threshold shape difference. V4 lesions caused profound, retinotopic, and apparently permanent disruptions of discrimination, regardless of whether the images represented single or multiple viewpoints. In V4 lesioned portions of the visual field, monkeys could discriminate objects only when they differed much more grossly in shape than was true in control locations. These effects of the lesion were virtually identical for discriminations that had been learned before lesions were placed and for those learned afterwards. As in previous studies, V4 lesions elevated contrast thresholds by approximately a factor of two, but control observations showed that this was not the basis of the disruption of shape discrimination. Manipulation of cues to shape showed that in control locations, monkeys maintained excellent shape discrimination despite a variety of stimulus alterations, whereas in V4 lesioned areas their performance was easily disrupted. This finding suggests that V4 may support visual shape discriminations by facilitating the use of multiple visual cues. However, the fact that single-viewpoint and multiple-viewpoint discriminations were similarly affected indicates that the disruption was not specific to 3D shape discrimination, but may apply to a variety of subtle discriminations.

Basic visual capacities and shape discrimination after lesions of extrastriate area V4 in macaques.

Ibotenic acid lesions were made in four macaque monkeys in a region of cortical area V4 that corresponds to the lower quadrant of one hemifield. For visual testing, fixation locus was monitored with scleral search coils and controlled behaviorally to place test stimuli either in the lesioned quadrant or in a control location in the opposite hemifield. Some basic visual capacities were slightly altered by the lesions; there was a two-fold reduction of luminance contrast sensitivity as well as red-green chromatic contrast sensitivity, both tested with stationary gratings. On the other hand, little or no loss was found when contrast sensitivity for detection or direction discrimination was tested with 10-Hz drifting gratings nor was there a reliable change in visual acuity. Hue and luminance matching were tested with a spatially more complex matching-to-sample task, but monkeys could not learn this task in the visual field locus of a V4 lesion. If previously trained at this locus, performance was not affected by the lesion. In contrast to the small effects on basic visual capabilities, performance on two form discrimination tasks was devastated by V4 lesions. The first involved discriminating the orientation of colinear groups of dots on a background of randomly placed dots. The second involved discriminating the orientation of a group of three line segments surrounded by differently oriented line segments. Some selectivity of the deficits for form discrimination was shown by the lack of an effect of the lesions on a global motion discrimination. These results show that while V4 lesions cause only slight disruptions of basic visual capacities, they profoundly disrupt form discriminations.

Motion perception following lesions of the superior temporal sulcus in the monkey.

We examined the effect of bilateral ibotenic acid lesions, aimed at areas MT/MST in three macaques, on their perception of motion. The medial boundary of the lesions in the three monkeys was near the dorsal end of the STS, but the lesions extended different lengths ventrally along the STS. The lesions extended the shortest distance ventrally monkeys 1 and 2, covering most of MST but possibly sparing a portion of lateral MT. That in monkey 3 damaged all of MT and MST bilaterally and extended through most of FST. All three lesions caused a temporary disruption, followed by at least partial recovery, of most motion thresholds. Permanent effects of the lesions on visual sensitivity were graded with lesion extent. Contrast sensitivity for detecting low-spatial-frequency (1 cycle/degree) drifting gratings over a wide range of drift rates, as well as for identifying their direction of motion, was slightly affected only in monkey 3. Only monkeys 2 and 3 showed a deficit in discriminating stimulus speed, and the size of the loss was two- to fourfold. Discrimination of opposite directions of dot pattern motion, which required integration of local motion signals, was mildly affected in monkeys 2 and 3, and not affected in monkey 1. However, addition of directional noise to this discrimination caused the performance of all monkeys to be permanently disrupted, especially that of monkeys 2 and 3. Finally, direction difference thresholds were elevated by a factor of 2-4 after the lesions in all three monkeys. Many of these deficits were more pronounced during the first 2 months of testing following the lesion. Thus, our results demonstrate that areas within dorsal STS make an important contribution to the performance of various motion perception tasks including the discrimination of small differences in direction and speed, and the perception of global motion in the presence of directional noise. The residual motion perception, even in the monkey with virtually complete removal of areas MT/MST, may suggest either that these tasks are normally mediated in part by cortical areas outside of areas MT and MST, or that the disrupted functions were partially assumed by other cortical areas after lesions.

Visual effects of lesions of cortical area V2 in macaques.

Ibotenic acid lesions were placed in two monkeys in a portion of cortical area V2 that corresponds to a lower quadrant of the visual field extending approximately 3-7 degrees from the fovea. For purposes of comparison, another lesion was placed in area V1 in one animal. A wide range of visual capacities were then measured, using a discrimination between vertical and horizontal orientation, in and near the affected regions of the visual field. Visual acuity declined sharply as the test stimulus approached the visual field location corresponding to the V1 lesion, and no threshold could be measured at its center. In contrast, lesions of area V2 caused no measurable decrease in acuity, nor was there any substantial effect on several measures of contrast sensitivity. Subsequently, two types of more complex visual discriminations were measured (also using a vertical-horizontal discrimination), and these discriminations were severely disrupted by V2 lesions. The first discrimination was of the orientation of two parallel lines of five colinear dots each. We measured the number of background dots that would bring the discrimination to threshold, and this number of dots was greatly decreased by a V2 lesion. The second discrimination was of the orientation of a group of three distinctive texture elements embedded in a six by six element texture. This task could not be done in the visual field region affected by the V2 lesion when the distinctive elements differed in orientation from the others. Control experiments showed that the discrimination could be done when the three distinctive elements differed in size or color. These results suggest that cortical area V2 is not needed for some low-level discriminations, but may be essential for tasks involving complex spatial discriminations.

Visual effects of damage to P ganglion cells in macaques.

Four indices of visual performance were measured in control macaques and in macaques that had been exposed to monomeric acrylamide, a neurotoxicant that preferentially damages P retinal ganglion cells. Morphological examination of the retina and visual pathways of these monkeys showed virtually complete loss of P ganglion cells over a region extending to at least 40 deg from the fovea, and relative sparing of M ganglion cells. The four tests examined visual functions for which the visual pathway from P ganglion cells might be of great importance: visual acuity, contrast discrimination, hyperacuity, and shape discrimination. In the acrylamide-dosed monkeys, visual acuity was reduced slightly more than fourfold, a somewhat larger reduction than that seen previously after ibotenic-acid lesions of the P pathway in the geniculate. The residual acuity was in good agreement with the Nyquist frequency calculated from the density of ON or OFF M ganglion cells. Contrast increment thresholds were elevated for the dosed monkeys only in one of the two conditions tested. The elevation was found only under those spatiotemporal conditions for which we have previously shown that contrast thresholds are increased by acrylamide exposure, and was most marked at low background contrasts. Vernier acuity was elevated in one dosed monkey, but not affected in a second monkey that also had severe loss of P ganglion cells. Finally, we found no effect of acrylamide exposure on the number of training trials required to learn simple or complex shape discriminations. These results support previous findings in showing that the P pathway mediates visual acuity, and they show that several other important aspects of visual perception are not exclusively dependent on the P pathway.

Does primate motion perception depend on the magnocellular pathway?

This study examined the importance of the primate magnocellular retinocortical pathway in the perception of moving stimuli. A portion of the magnocellular pathway was permanently and selectively interrupted by ibotenic acid injections in the LGN of macaque monkeys. We then tested contrast sensitivity for detecting moving stimuli, as well as two indices of motion perception, contrast sensitivity for opposite direction discrimination and speed difference thresholds, in the affected portion of the visual field. Magnocellular lesions greatly reduced detection contrast sensitivity at high temporal and low spatial frequencies and had a similar effect on contrast sensitivity for opposite direction discrimination under these same stimulus conditions. Consequently, opposite direction discriminations could be made at contrast threshold, suggesting that magnocellular lesions reduced the visibility of stimuli used to test direction perception, but did not act directly on direction perception. Magnocellular lesions also elevated speed difference thresholds under some stimulus conditions. However, this deficit was reduced or eliminated by raising the contrast of the test stimulus. Together, these findings suggest that magnocellular lesions reduce the visibility of stimuli used to test motion perception but that they do not appear to alter motion perception otherwise.

The effects of parvocellular lateral geniculate lesions on the acuity and contrast sensitivity of macaque monkeys.

The effects of ablating the visual pathway that passes through the parvocellular (dorsal) LGN were tested in 2 macaque monkeys by measuring acuity and both luminance and chromatic contrast sensitivity. Thresholds were tested monocularly before and after ibotenic acid was used to lesion parvocellular layers 4 and 6 of the contralateral geniculate. The injections were centered at the representation of 6 degrees in the temporal field on the horizontal meridian, and vision was tested with localized stimuli at this location. In addition, in one of the monkeys, a lesion was made in magnocellular layer 1 of the opposite geniculate, and the same thresholds were tested. Physiological and anatomical reconstructions demonstrated complete destruction of the target layers in 1 parvocellular lesions and in the magnocellular lesion, and sparing of the nontarget layers in the tested region. Parvocellular lesions caused a 3-4-fold reduction in visual acuity within the affected part of the visual field, while the magnocellular lesion did not affect acuity. Both luminance and chromatic contrast sensitivity, tested with stationary gratings of 2 c/degree, were severely reduced by parvocellular lesions, but not affected by the magnocellular lesion. However, when luminance contrast sensitivity was tested with 1 c/degree gratings, counterphase modulated at 10 Hz, it was reduced by both parvocellular and magnocellular lesions. This study demonstrates that the parvocellular pathway dominates chromatic vision, acuity, and contrast detection at low temporal and high spatial frequencies, while the magnocellular pathway may mediate contrast detection at higher temporal and lower spatial frequencies.

Macaque vision after magnocellular lateral geniculate lesions.

Ibotenic-acid lesions of the magnocellular portion of the macaque lateral geniculate nucleus were used to examine the role of the M-cell pathway in spatio-temporal contrast sensitivity. A lesion was placed in layer 1 of the lateral geniculate of each of two monkeys. Physiological mapping in one animal demonstrated that the visual-field locus of the lesion was on the horizontal meridian, approximately 6 deg in the temporal field. Visual thresholds were tested monocularly in the contralateral eye, and fixation locus was monitored with a scleral search coil to control the retinal location of the test target. Three threshold measures were clearly disrupted by the magnocellular lesions. Contrast sensitivity for a 1 cycle/deg grating that drifted at 10 Hz was reduced from about twofold greater than, to about the same as, that for 10-Hz counterphase modulated gratings. Sensitivity for a very low spatial frequency (Gaussian blob), 10-Hz flickering stimulus was reduced so severely that no threshold could be measured. In addition, flicker resolution was greatly reduced at lower modulation depths (0.22), but not at higher depths (1.0). Two of the measured thresholds were unaffected by the lesions. Contrast sensitivity for 2 cycle/deg stationary gratings remained intact, and little or no effect on sensitivity was found for 1 cycle/deg, 10-Hz counterphase modulated gratings. Together, these results suggest that the magnocellular pathway makes little contribution to visual sensitivity at low to moderate temporal frequencies. On the other hand, some contribution to detection sensitivity is evident at lower spatial and high temporal frequencies, especially for drifting stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial resolution across the macaque retina.

Grating acuity was measured as a function of eccentricity from the fovea in two macaques. A vertical-horizontal orientation discrimination was used to determine acuity, and the retinal locus of the test grating was controlled by training them to fixate a spot placed at various distances from the stimulus. Their head was fixed in place and fixation was monitored with a scleral search coil. The acuity of monkeys across the retina was similar to that previously measured in human subjects, reaching a peak of about 38 c/deg at the fovea, and decreasing about 10-fold by 30 deg eccentricity. Acuity was slightly higher in the temporal than in the nasal visual field. The shape of the acuity-eccentricity function suggested a dependence on cone density near the fovea, and on the density of P ganglion cells at eccentricities beyond 10 deg. Existing physiological data suggest the possibility that macaque acuity may also be limited in part by spatial averaging across the receptive field of retinal ganglion cells.

Selective degeneration of the parvocellular-projecting retinal ganglion cells in a New World monkey, Saimiri sciureus.

Selective degeneration of retinal ganglion cells projecting to parvocellular layers of the dorsal lateral geniculate nucleus (LGN) was observed in squirrel monkeys (Saimiri sciureus) exposed to a range of doses of acrylamide monomer. Similar acrylamide-induced neuronal loss has previously been reported in parvocellular-projecting ganglion cells of macaques, but no such selective degeneration has been found in acrylamide-dosed rats, squirrels, rabbits or cats. The extent of ganglion cell loss observed in the present study suggests that in the squirrel monkey, as in the macaque, a majority of ganglion cells project to parvocellular layers of the LGN. The locus of optic tract degeneration suggests that the squirrel monkey parvocellular pathway passes in dorsolateral optic tract, as does that of the macaque. Patterns of decreases in cytochrome oxidase activity confirm that, in both of these primates, geniculocortical pathways driven by these vulnerable neurons project to cortical layers 4A and 4C beta. These results suggest close parallels in the neuroanatomical projections and toxic vulnerability of the parvocellular-projecting pathway in New and Old World monkeys. They indicate that acrylamide intoxication can be used to selectively damage this pathway in order to study the functional roles of parallel visual pathways in both New and Old World monkeys.

Subchronic dosing of macaques with 2,5-hexanedione causes long-lasting motor dysfunction but reversible visual loss.

Visual sensitivity and neurological status were monitored in four female macaque monkeys dosed orally with 2.5-hexanedione (0.64 mM/kg, 5 days per week) for 15 or 17 weeks. The first sign of toxicity was intention tremor seen after 3 months of dosing. This was followed, a week later, by a moderate decrease in visual contrast sensitivity, which returned to baseline 6 to 11 weeks after the end of dosing. Acuity and flicker resolution were not disrupted. A progressive general weakness ensued for 5 to 7 weeks after dosing had ended. Some slow recovery was seen, although the animals remained severely disabled 20 weeks after dosing was discontinued. Pathologic changes were examined 3 weeks (one monkey) and 20 weeks (three monkeys) after dosing. Soon after the end of dosing, axonal swellings were present throughout the distal optic tracts, peripheral nerves, and long tracts of the spinal cord. Twenty weeks after dosing, there was no indication of degeneration in the retinocortical pathway. Peripheral nerves showed widespread axonal loss, residual ongoing degeneration, and only slight regeneration. Axon loss and gliosis were evident in distal dorsal columns, and to a lesser extent, dorsal spinocerebellar and corticospinal tracts. These effects of 2,5-hexanedione on the macaque differ from those found previously for two other axonotoxic compounds, acrylamide monomer and carbon disulfide, which caused marked permanent degeneration that was most prominent in the visual system.

Chromatic and achromatic vision of macaques: role of the P pathway.

Chromatic and achromatic contrast sensitivity were measured in a human observer, 2 normal macaque monkeys, and 3 monkeys with severe toxicant-induced damage to the parvocellular projecting retinogeniculate pathway (P cell-deficient monkeys). Damage to the P pathway was produced by the oral administration of acrylamide monomer (Eskin and Merigan, 1986). Contrast sensitivity was measured in all subjects with isochromatic luminance gratings, as well as isoluminant chromatic gratings, modulated along several directions of a color space that represents color-opponent and luminance contrast (Krauskopf et al., 1986). The chromatic and achromatic sensitivity of the control monkeys was virtually identical to that of the human observer. Chromatic sensitivity of the P cell-deficient monkeys, measured at a low spatial frequency (0.3 c/deg), along a constant-blue color axis, was 0.9-1.5 log units lower than that of controls. Similar losses were seen along a tritanopic confusion axis and along 2 intermediate axes of color direction. Chromatic thresholds measured at higher spatial frequency (2.0 c/deg) were similarly reduced. Counterphase-modulated chromatic gratings were used to test color sensitivity over a range of temporal frequencies up to 15 Hz, and the loss of color vision was substantial over the entire range of frequencies. The luminance contrast sensitivity of the P cell-deficient monkeys for stationary gratings decreased after exposure by 0.5-0.8 log units. These results indicate that the chromatic and achromatic spatial vision of macaques is very similar to that of humans. They also suggest that the P pathway plays an important role in macaque chromatic sensitivity at all spatial frequencies, as well as achromatic sensitivity at high spatial and lower temporal frequencies.