Life Cycle and Lensing of a Macular Microcyst.

INTRODUCTION: Microscopic details about retinal conditions can provide insight into pathological mechanisms, but these are ordinarily difficult to obtain in situ. We demonstrate how high-resolution imaging and optical modeling can be combined to reveal morphological features of a macular microcyst, offering insight into microcyst formation. OBJECTIVE: To use adaptive optics scanning laser ophthalmoscopic (AOSLO) images to track a transient retinal microcyst and derive its 3-dimensional shape. METHODS: A series of AOSLO images were gathered before, during, and after a transient retinal microcyst developed in an otherwise normal healthy 26-year-old male subject. Optical coherence tomography (OCT) independently confirmed the location of the microcyst. Optical modeling was conducted to quantify the lensing effect of the optically uniform microcyst and to determine its 3-dimensional shape. Increment threshold sensitivity, targeted within and around the microcyst, was tested to see if cone photoreceptor function was affected. RESULTS: A transient microcyst appeared as a 50 µm diameter circle in AOSLO images, localized to the inner nuclear layer. Based on image distortion of the photoreceptor mosaic, optical modeling suggests that the microcyst had the shape of an aspherical lens, distinguishable from a spherical, cylindrical, or elliptical shape, indicative of an edematous expansion of laminar tissue. The microcyst spontaneously resolved about 30 days after first discovery. No changes to the photoreceptor mosaic ensued from the presence of the microcyst, and functional testing of the photoreceptors below the microcyst indicated no loss of light sensitivity. CONCLUSIONS: Microcysts have been associated with numerous subtypes of optic nerve degeneration, including multiple sclerosis and various inherited neuropathies. This microcyst appeared in a healthy individual and resolved without intervention. Lensing effects can be used to determine microcyst shape, which cannot be resolved by OCT imaging, and to help infer etiology.

Light reflectivity and interference in cone photoreceptors.

In several modes of retinal imaging, the primary means of visualizing cone photoreceptors is from reflected light. Understanding how such images are formed, particularly when adaptive optics techniques are used, will help to guide their interpretation. Toward this end, we used finite difference beam propagation to model reflections from cone photoreceptors. We investigated the formation of cone images in adaptive optics scanning laser ophthalmoscopy (AOSLO) and optical coherence tomography (AOOCT). Three cone models were tested, one made up of three segments of varying refractive index, the other two having additional boundaries at the inner/outer segment junction and outer segment tip. Images formed by the first model did not correspond to AOOCT observations in the literature, while the latter two did. The predicted distributions of reflected light intensity from the latter cone models were compared to the distribution from AOSLO images, both studied with light sources of varied coherence length. The cone model with the most reflections at the inner/outer segment junction best fit the data measured in vivo. These results show that variance in cone reflection can originate from light interfering from reflectors much more closely spaced than the outer segment length. We also show that subtracting images taken with different coherence length sources highlights these changes in interference. Differential coherence images of cones occasionally revealed an annular reflection profile, which modeling showed to be very sensitive to cone size and the gaps bracketing the outer segment, suggesting that such imaging may be useful for probing photoreceptor morphology.

Light propagation and capture in cone photoreceptors.

The light capturing properties of cone photoreceptors create the elementary signals that form the basis of vision. Variation in the amplitude of individual cone signals has been found physiologically as part of normal retinal circuit processing. Less well characterized is how cone signals may vary due to purely optical properties. We present a model of light propagation in cones using a finite difference beam propagation method to simulate how light from a small stimulus travels through a cone plus its immediate neighbors. The model calculates the amount of light absorbed in the cone outer segments, from which an estimate of the photoresponse can be made. We apply the method to adaptive optics microstimulation to find the optimum optical conditions that will confine the most light into a single cone in the human retina. We found that light capture is especially sensitive to beam size at the pupil and to the cone diameter itself, with the two factors having a complex relationship leading to sizable variation in light capture. Model predictions were validated with two types of psychophysical data. The model can be employed with arbitrary stimuli and photoreceptor parameters, making it a useful tool for studying photoreceptor function in normal or diseased conditions.

Spatiochromatic Interactions between Individual Cone Photoreceptors in the Human Retina.

A remarkable feature of human vision is that the retina and brain have evolved circuitry to extract useful spatial and spectral information from signals originating in a photoreceptor mosaic with trichromatic constituents that vary widely in their relative numbers and local spatial configurations. A critical early transformation applied to cone signals is horizontal-cell-mediated lateral inhibition, which imparts a spatially antagonistic surround to individual cone receptive fields, a signature inherited by downstream neurons and implicated in color signaling. In the peripheral retina, the functional connectivity of cone inputs to the circuitry that mediates lateral inhibition is not cone-type specific, but whether these wiring schemes are maintained closer to the fovea remains unsettled, in part because central retinal anatomy is not easily amenable to direct physiological assessment. Here, we demonstrate how the precise topography of the long (L)-, middle (M)-, and short (S)-wavelength-sensitive cones in the human parafovea (1.5° eccentricity) shapes perceptual sensitivity. We used adaptive optics microstimulation to measure psychophysical detection thresholds from individual cones with spectral types that had been classified independently by absorptance imaging. Measured against chromatic adapting backgrounds, the sensitivities of L and M cones were, on average, receptor-type specific, but individual cone thresholds varied systematically with the number of preferentially activated cones in the immediate neighborhood. The spatial and spectral patterns of these interactions suggest that interneurons mediating lateral inhibition in the central retina, likely horizontal cells, establish functional connections with L and M cones indiscriminately, implying that the cone-selective circuitry supporting red-green color vision emerges after the first retinal synapse.SIGNIFICANCE STATEMENT We present evidence for spatially antagonistic interactions between individual, spectrally typed cones in the central retina of human observers using adaptive optics. Using chromatic adapting fields to modulate the relative steady-state activity of long (L)- and middle (M)-wavelength-sensitive cones, we found that single-cone detection thresholds varied predictably with the spectral demographics of the surrounding cones. The spatial scale and spectral pattern of these photoreceptor interactions were consistent with lateral inhibition mediated by retinal horizontal cells that receive nonselective input from L and M cones. These results demonstrate a clear link between the neural architecture of the visual system inputs-cone photoreceptors-and visual perception and have implications for the neural locus of the cone-specific circuitry supporting color vision.

Vision science and adaptive optics, the state of the field.

Adaptive optics is a relatively new field, yet it is spreading rapidly and allows new questions to be asked about how the visual system is organized. The editors of this feature issue have posed a series of question to scientists involved in using adaptive optics in vision science. The questions are focused on three main areas. In the first we investigate the use of adaptive optics for psychophysical measurements of visual system function and for improving the optics of the eye. In the second, we look at the applications and impact of adaptive optics on retinal imaging and its promise for basic and applied research. In the third, we explore how adaptive optics is being used to improve our understanding of the neurophysiology of the visual system.

Normal Perceptual Sensitivity Arising From Weakly Reflective Cone Photoreceptors.

PURPOSE: To determine the light sensitivity of poorly reflective cones observed in retinas of normal subjects, and to establish a relationship between cone reflectivity and perceptual threshold. METHODS: Five subjects (four male, one female) with normal vision were imaged longitudinally (7-26 imaging sessions, representing 82-896 days) using adaptive optics scanning laser ophthalmoscopy (AOSLO) to monitor cone reflectance. Ten cones with unusually low reflectivity, as well as 10 normally reflective cones serving as controls, were targeted for perceptual testing. Cone-sized stimuli were delivered to the targeted cones and luminance increment thresholds were quantified. Thresholds were measured three to five times per session for each cone in the 10 pairs, all located 2.2 to 3.3° from the center of gaze. RESULTS: Compared with other cones in the same retinal area, three of 10 monitored dark cones were persistently poorly reflective, while seven occasionally manifested normal reflectance. Tested psychophysically, all 10 dark cones had thresholds comparable with those from normally reflecting cones measured concurrently (P = 0.49). The variation observed in dark cone thresholds also matched the wide variation seen in a large population (n = 56 cone pairs, six subjects) of normal cones; in the latter, no correlation was found between cone reflectivity and threshold (P = 0.0502). CONCLUSIONS: Low cone reflectance cannot be used as a reliable indicator of cone sensitivity to light in normal retinas. To improve assessment of early retinal pathology, other diagnostic criteria should be employed along with imaging and cone-based microperimetry.

Mapping the perceptual grain of the human retina.

In humans, experimental access to single sensory receptors is difficult to achieve, yet it is crucial for learning how the signals arising from each receptor are transformed into perception. By combining adaptive optics microstimulation with high-speed eye tracking, we show that retinal function can be probed at the level of the individual cone photoreceptor in living eyes. Classical psychometric functions were obtained from cone-sized microstimuli targeted to single photoreceptors. Revealed psychophysically, the cone mosaic also manifests a variable sensitivity to light across its surface that accords with a simple model of cone light capture. Because this microscopic grain of vision could be detected on the perceptual level, it suggests that photoreceptors can act individually to shape perception, if the normally suboptimal relay of light by the eye's optics is corrected. Thus the precise arrangement of cones and the exact placement of stimuli onto those cones create the initial retinal limits on signals mediating spatial vision.

Cortical metabolic activity matches the pattern of visual suppression in strabismus.

When an eye becomes deviated in early childhood, a person does not experience double vision, although the globes are aimed at different targets. The extra image is prevented from reaching perception in subjects with alternating exotropia by suppression of each eye's peripheral temporal retina. To test the impact of visual suppression on neuronal activity in primary (striate) visual cortex, the pattern of cytochrome oxidase (CO) staining was examined in four macaques raised with exotropia by disinserting the medial rectus muscles shortly following birth. No ocular dominance columns were visible in opercular cortex, where the central visual field is represented, indicating that signals coming from the central retina in each eye were perceived. However, the border strips at the edges of ocular dominance columns appeared pale, reflecting a loss of activity in binocular cells from disruption of fusion. In calcarine cortex, where the peripheral visual field is represented, there were alternating pale and dark bands resembling ocular dominance columns. To interpret the CO staining pattern, [(3)H]proline was injected into the right eye in two monkeys. In the right calcarine cortex, the pale CO columns matched the labeled proline columns of the right eye. In the left calcarine cortex, the pale CO columns overlapped the unlabeled columns of the left eye in the autoradiograph. Therefore, metabolic activity was reduced in the ipsilateral eye's ocular dominance columns which serve peripheral temporal retina, in a fashion consistent with the topographic organization of suppression scotomas in humans with exotropia.

Measurement and correction of transverse chromatic offsets for multi-wavelength retinal microscopy in the living eye.

A special challenge arises when pursuing multi-wavelength imaging of retinal tissue in vivo, because the eye's optics must be used as the main focusing elements, and they introduce significant chromatic dispersion. Here we present an image-based method to measure and correct for the eye's transverse chromatic aberrations rapidly, non-invasively, and with high precision. We validate the technique against hyperacute psychophysical performance and the standard chromatic human eye model. In vivo correction of chromatic dispersion will enable confocal multi-wavelength images of the living retina to be aligned, and allow targeted chromatic stimulation of the photoreceptor mosaic to be performed accurately with sub-cellular resolution.

Neuronal projections from V1 to V2 in amblyopia.

The mechanism of amblyopia in children with congenital cataract is not understood fully, but studies in macaques have shown that geniculate synapses are lost in striate cortex (V1). To search for other projection abnormalities in amblyopia, the pathway from V1 to V2 was examined using a triple-label technique in three animals raised with monocular suture. [(3)H]proline was injected into one eye to label the ocular dominance columns. Cholera toxin B subunit conjugated to gold (CTB-Au) was injected into V2 to label V1 projection neurons. Alternate sections were processed for cytochrome oxidase (CO) and CTB-Au, or dipped for autoradiography. Eight fields of CTB-Au-labeled cells in V1 opposite injection sites were plotted in layers 2/3 or 4B. After thin stripe injection, labeled cells were concentrated in CO patches. Despite column shrinkage, cells in deprived and normal columns were equal in size and density in both layers 2/3 and 4B. After pale or thick stripe injection, labeled cells were concentrated in interpatches. Only 23% of projection neurons originated from deprived columns. This reduction exceeded the degree of column shrinkage, a result explained by the fact that column shrinkage causes disproportionate loss of interpatch territory. These data indicate that early monocular form deprivation does not alter the segregation of patch and interpatch pathways to V2 stripes or cause selective loss or atrophy of V1 projection neurons. The effect of shrinkage of geniculocortical afferents in layer 4C following visual deprivation is not amplified further by attenuation of the amblyopic eye's projections from V1 to V2.

Second order dimensionality reduction using minimum and maximum mutual information models.

Conventional methods used to characterize multidimensional neural feature selectivity, such as spike-triggered covariance (STC) or maximally informative dimensions (MID), are limited to Gaussian stimuli or are only able to identify a small number of features due to the curse of dimensionality. To overcome these issues, we propose two new dimensionality reduction methods that use minimum and maximum information models. These methods are information theoretic extensions of STC that can be used with non-Gaussian stimulus distributions to find relevant linear subspaces of arbitrary dimensionality. We compare these new methods to the conventional methods in two ways: with biologically-inspired simulated neurons responding to natural images and with recordings from macaque retinal and thalamic cells responding to naturalistic time-varying stimuli. With non-Gaussian stimuli, the minimum and maximum information methods significantly outperform STC in all cases, whereas MID performs best in the regime of low dimensional feature spaces.

Orientation tuning of cytochrome oxidase patches in macaque primary visual cortex.

The abundant concentration of cytochrome oxidase in patches or blobs of primate striate cortex has never been explained. Patches are thought to contain unoriented, color-opponent neurons. Lacking orientation selectivity, these cells might endow patches with high metabolic activity because they respond to all contours in visual scenes. To test this idea, we measured orientation tuning in layer 2/3 of macaque cortical area V1 using acutely implanted 100-electrode arrays. Each electrode recording site was identified and assigned to the patch or interpatch compartment. The mean orientation bandwidth of cells was 28.4° in patches and 25.8° in interpatches. Neurons in patches were indeed less orientation selective, but the difference was subtle, indicating that the processing of form and color is not strictly segregated in V1. The most conspicuous finding was that patch cells had a 49% greater overall firing rate. This global difference in neuronal responsiveness, rather than an absence of orientation tuning, may account for the rich mitochondrial enzyme activity that defines patches.

Minimal models of multidimensional computations.

The multidimensional computations performed by many biological systems are often characterized with limited information about the correlations between inputs and outputs. Given this limitation, our approach is to construct the maximum noise entropy response function of the system, leading to a closed-form and minimally biased model consistent with a given set of constraints on the input/output moments; the result is equivalent to conditional random field models from machine learning. For systems with binary outputs, such as neurons encoding sensory stimuli, the maximum noise entropy models are logistic functions whose arguments depend on the constraints. A constraint on the average output turns the binary maximum noise entropy models into minimum mutual information models, allowing for the calculation of the information content of the constraints and an information theoretic characterization of the system's computations. We use this approach to analyze the nonlinear input/output functions in macaque retina and thalamus; although these systems have been previously shown to be responsive to two input dimensions, the functional form of the response function in this reduced space had not been unambiguously identified. A second order model based on the logistic function is found to be both necessary and sufficient to accurately describe the neural responses to naturalistic stimuli, accounting for an average of 93% of the mutual information with a small number of parameters. Thus, despite the fact that the stimulus is highly non-Gaussian, the vast majority of the information in the neural responses is related to first and second order correlations. Our results suggest a principled and unbiased way to model multidimensional computations and determine the statistics of the inputs that are being encoded in the outputs.

V1 interpatch projections to v2 thick stripes and pale stripes.

Cytochrome oxidase (CO) reveals two compartments in V1 (patches and interpatches) and three compartments in V2 (thin, pale, and thick stripes). Previously, it was shown that thin stripes receive input predominantly from patches. Here we examined the projections to thick and pale stripes in macaques, revealed by retrograde tracer injections. After thick stripe injection, cells were distributed in layer 2/3 (67%), layer 4A (7%), layer 4B (23%), and layer 5/6 (2%). Except in layer 5/6, cells were concentrated in interpatches, with a stronger bias in layer 2/3 (84%) than in layer 4B (75%). After pale stripe injection, cells were found in layer 2/3 (87%), layer 4A (2%), layer 4B (10%), and layer 5/6 (2%). As for thick stripes, cells were located preferentially in interpatches in layer 2/3 (84%) and layer 4B (72%) but not in layer 5/6. Thick stripes received a higher proportion of their input from layer 4B, compared with pale stripes, consistent with reports that thick stripe neurons exhibit a pronounced layer 4B influence. This difference aside, both stripe types receive similar inputs from V1, at least in terms of cortical layer and CO compartment. This finding was bolstered by injecting different tracers into pale and thick stripes; 10-27% of cells were double labeled, with most located in interpatches. These results suggest that the distinctive receptive field properties of neurons in thick and pale stripes are generated by local V2 circuits, or by other specific projections, rather than by differing sources of laminar and compartmental input from V1.

Resolving single cone inputs to visual receptive fields.

With the current techniques available for mapping receptive fields, it is impossible to resolve the contribution of single cone photoreceptors to the response of central visual neurons. Using adaptive optics to correct for ocular aberrations, we delivered micron-scale spots of light to the receptive field centers of neurons in the macaque lateral geniculate nucleus. Parvocellular LGN neurons mapped in this manner responded with high reliability to stimulation of single cones.

Preserving information in neural transmission.

Along most neural pathways, the spike trains transmitted from one neuron to the next are altered. In the process, neurons can either achieve a more efficient stimulus representation, or extract some biologically important stimulus parameter, or succeed at both. We recorded the inputs from single retinal ganglion cells and the outputs from connected lateral geniculate neurons in the macaque to examine how visual signals are relayed from retina to cortex. We found that geniculate neurons re-encoded multiple temporal stimulus features to yield output spikes that carried more information about stimuli than was available in each input spike. The coding transformation of some relay neurons occurred with no decrement in information rate, despite output spike rates that averaged half the input spike rates. This preservation of transmitted information was achieved by the short-term summation of inputs that geniculate neurons require to spike. A reduced model of the retinal and geniculate visual responses, based on two stimulus features and their associated nonlinearities, could account for >85% of the total information available in the spike trains and the preserved information transmission. These results apply to neurons operating on a single time-varying input, suggesting that synaptic temporal integration can alter the temporal receptive field properties to create a more efficient representation of visual signals in the thalamus than the retina.

Complete pattern of ocular dominance columns in human primary visual cortex.

The occipital lobes were obtained after death from six adult subjects with monocular visual loss. Flat-mounts were processed for cytochrome oxidase (CO) to reveal metabolic activity in the primary (V1) and secondary (V2) visual cortices. Mean V1 surface area was 2643 mm2 (range, 1986-3477 mm2). Ocular dominance columns were present in all cases, having a mean width of 863 microm. There were 78-126 column pairs along the V1 perimeter. Human column patterns were highly variable, but in at least one person they resembled a scaled-up version of macaque columns. CO patches in the upper layers were centered on ocular dominance columns in layer 4C, with one exception. In this individual, the columns in a local area resembled those present in the squirrel monkey, and no evidence was found for column/patch alignment. In every subject, the blind spot of the contralateral eye was conspicuous as an oval region without ocular dominance columns. It provided a precise landmark for delineating the central 15 degrees of the visual field. A mean of 53.1% of striate cortex was devoted to the representation of the central 15 degrees. This fraction was less than the proportion of striate cortex allocated to the representation of the central 15 degrees in the macaque. Within the central 15 degrees, each eye occupied an equal territory. Beyond this eccentricity, the contralateral eye predominated, occupying 63% of the cortex. In one subject, monocular visual loss began at age 4 months, causing shrinkage of ocular dominance columns. In V2, which had a larger surface area than V1, CO stripes were present but could not be classified as thick or thin.

Transmission of spike trains at the retinogeniculate synapse.

Retinal spikes impinging on relay neurons in the lateral geniculate nucleus (LGN) generate synaptic potentials, which sometimes produce spikes sent to visual cortex. We examined how signal transmission is regulated in the macaque LGN by recording the retinal input to a single LGN neuron while stimulating the receptive field center with a naturalistic luminance sequence. After extracting the EPSPs, which are often partially merged with spike waveforms, we found that >95% of spikes were associated with an EPSP from a single retinal ganglion cell. Each spike within a "burst" train was generated by an EPSP, indicating that LGN bursts are inherited from retinal bursts. LGN neurons rarely fired unless at least two EPSPs summated within 40 ms. This facilitation in EPSP efficacy was followed by depression. If a spike was generated by the first EPSP in a pair, it did not alter the efficacy of the second EPSP. Hence, the timing of EPSPs arising from the primary retinal driver governs synaptic efficacy and provides the basis for successful retinogeniculate transmission.

Thalamic filtering of retinal spike trains by postsynaptic summation.

At many synapses in the central nervous system, spikes within high-frequency trains have a better chance of driving the postsynaptic neuron than spikes occurring in isolation. We asked what mechanism accounts for this selectivity at the retinogeniculate synapse. The amplitude of synaptic potentials was remarkably constant, ruling out a major role for presynaptic mechanisms such as synaptic facilitation. Instead, geniculate spike trains could be predicted from retinal spike trains on the basis of postsynaptic summation. This simple form of integration explains the response differences between a geniculate neuron and its main retinal driver, and thereby determines the flow of visual information to cortex.

Neurons in V1 patch columns project to V2 thin stripes.

In the primate, connections between primary visual cortex (V1) and the second visual area (V2) are segregated according to the characteristic pattern of cytochrome oxidase (CO) activity in each of these cortical areas. Patches supply thin stripes, whereas interpatches supply pale stripes and thick stripes. Previously, the projection from patches to thin stripes was reported to arise exclusively from layer 2/3. In this present report, we made injections of a retrograde tracer, cholera toxin-B (CTB-Au), into macaque V2 thin stripes to re-examine the laminar origin of their input from V1. While the great majority of cells indeed resided in layer 2/3, small populations were also present in layers 4A, 4B, and 5/6. The location of CTB-filled cells in each layer was analyzed to determine the relationship with CO patches. Cells in layers 2/3, 4A, and 4B were aggregated into patches, forming columns that project to thin stripes. Surprisingly, cells in layer 5/6 were scattered, seemingly at random. These findings confirm that the main V1 projection to V2 stripes emanates from patches in layer 2/3. However, multiple V1 layers innervate V2 thin stripes, and the projection from layer 5/6 does not respect the patch/interpatch dichotomy.