Electroretinographic responses to periodic stimuli in primates and the relevance for visual perception and for clinical studies
Currently, electroretinograms (ERGs) are mainly recorded while using flashes as stimuli. In this review, we will argue that strong flashes are not ideal for studying visual information processing. ERG responses to periodic stimuli may be more strongly associated with the activity of post-receptoral neurons (belonging to different retino-geniculate pathways) and, therefore, be more relevant for visual perception. We will also argue that the use of periodic stimuli may be an attractive addition to clinically available retinal electrophysiological methods.
Synaptotagmin-9 in mouse retina
Synaptotagmin-9 (Syt9) is a Ca sensor mediating fast synaptic release expressed in various parts of the brain. The presence and role of Syt9 in retina is unknown. We found evidence for Syt9 expression throughout the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner. We crossed Syt9 mice with Rho-iCre, HRGP-Cre, and CMV-Cre mice to generate mice in which Syt9 was eliminated from rods (rod), cones (cone), or whole animals (CMV). CMV mice showed an increase in scotopic electroretinogram (ERG) b-waves evoked by bright flashes with no change in a-waves. Cone-driven photopic ERG b-waves were not significantly different in CMV knockout mice and selective elimination of Syt9 from cones had no effect on ERGs. However, selective elimination from rods decreased scotopic and photopic b-waves as well as oscillatory potentials. These changes occurred only with bright flashes where cone responses contribute. Synaptic release was measured in individual rods by recording anion currents activated by glutamate binding to presynaptic glutamate transporters. Loss of Syt9 from rods had no effect on spontaneous or depolarization-evoked release. Our data show that Syt9 acts at multiple sites in the retina and suggest that it may play a role in regulating transmission of cone signals by rods.
Chemically induced cone degeneration in the 13-lined ground squirrel
Animal models of retinal degeneration are critical for understanding disease and testing potential therapies. Inducing degeneration commonly involves the administration of chemicals that kill photoreceptors by disrupting metabolic pathways, signaling pathways, or protein synthesis. While chemically induced degeneration has been demonstrated in a variety of animals (mice, rats, rabbits, felines, 13-lined ground squirrels (13-LGS), pigs, chicks), few studies have used noninvasive high-resolution retinal imaging to monitor the cellular effects. Here, we used longitudinal scanning light ophthalmoscopy (SLO), optical coherence tomography, and adaptive optics SLO imaging in the euthermic, cone-dominant 13-LGS (46 animals, 52 eyes) to examine retinal structure following intravitreal injections of chemicals, which were previously shown to induce photoreceptor degeneration, throughout the active season of 2019 and 2020. We found that iodoacetic acid induced severe pan-retinal damage in all but one eye, which received the lowest concentration. While sodium nitroprusside successfully induced degeneration of the outer retinal layers, the results were variable, and damage was also observed in 50% of contralateral control eyes. Adenosine triphosphate and tunicamycin induced outer retinal specific damage with varying results, while eyes injected with thapsigargin did not show signs of degeneration. Given the variability of damage we observed, follow-up studies examining the possible physiological origins of this variability are critical. These additional studies should further advance the utility of chemically induced photoreceptor degeneration models in the cone-dominant 13-LGS.
Pre-stimulus bioelectrical activity in lightadapted ERG under blue versus white background - CORRIGENDUM
Pre-stimulus bioelectrical activity in light-adapted ERG under blue versus white background
To compare the baseline signal between two conditions used to generate the photopic negative response (PhNR) of the full-field electroretinogram (ERG): red flash on a blue background (RoB) and white flash on a white background (LA3). The secondary purpose is to identify how the level of pre-stimulus signal affects obtaining an unambiguous PhNR component. A retrospective chart review was conducted on four cohorts of patients undergoing routine ERG testing. In each group, LA3 was recorded the same way while RoB was generated differently using various luminances of red and blue light. The background bioelectrical activity 30 ms before the flash was extracted, and the root mean square (RMS) of the signal was calculated and compared between RoB and LA3 using Wilcoxon test. Pre-stimulus noise was significantly higher under RoB stimulation versus LA3 in all four conditions for both right and left eyes (ratio RoB/LA3 RMS 1.70 and 1.57 respectively, < 0.033). There was also no significant difference between the RMS of either LA3 or RoB across protocols, indicating that the baseline noise across cohorts were comparable. Additionally, pre-stimulus noise was higher in signals where PhNR was not clearly identifiable as an ERG component versus signals with the presence of unambiguous PhNR component under RoB in all four groups for both eyes ( < 0.05), whereas the difference under LA3 was less pronounced. Our study suggests that LA3 produces less background bioelectrical activity, likely due to decreased facial muscle activity. As it seems that the pre-stimulus signal level affects PhNR recordability, LA3 may also produce a better-quality signal compared to RoB. Therefore, until conditions for a comparable bioelectrical activity under RoB are established, we believe that LA3 should be considered at least as a supplementary method to evaluate retinal ganglion cell function by ERG.
Evolution of the visual system in ray-finned fishes
The vertebrate eye allows to capture an enormous amount of detail about the surrounding world which can only be exploited with sophisticated central information processing. Furthermore, vision is an active process due to head and eye movements that enables the animal to change the gaze and actively select objects to investigate in detail. The entire system requires a coordinated coevolution of its parts to work properly. Ray-finned fishes offer a unique opportunity to study the evolution of the visual system due to the high diversity in all of its parts. Here, we are bringing together information on retinal specializations (fovea), central visual centers (brain morphology studies), and eye movements in a large number of ray-finned fishes in a cladistic framework. The nucleus glomerulosus-inferior lobe system is well developed only in Acanthopterygii. A fovea, independent eye movements, and an enlargement of the nucleus glomerulosus-inferior lobe system coevolved at least five times independently within Acanthopterygii. This suggests that the nucleus glomerulosus-inferior lobe system is involved in advanced object recognition which is especially well developed in association with a fovea and independent eye movements. None of the non-Acanthopterygii have a fovea (except for some deep sea fish) or independent eye movements and they also lack important parts of the glomerulosus-inferior lobe system. This suggests that structures for advanced visual object recognition evolved within ray-finned fishes independent of the ones in tetrapods and non-ray-finned fishes as a result of a coevolution of retinal, central, and oculomotor structures.
Two mechanisms for direction selectivity in a model of the primate starburst amacrine cell
In a recent study, visual signals were recorded for the first time in starburst amacrine cells of the macaque retina, and, as for mouse and rabbit, a directional bias observed in calcium signals was recorded from near the dendritic tips. Stimulus motion from the soma toward the tip generated a larger calcium signal than motion from the tip toward the soma. Two mechanisms affecting the spatiotemporal summation of excitatory postsynaptic currents have been proposed to contribute to directional signaling at the dendritic tips of starbursts: (1) a "morphological" mechanism in which electrotonic propagation of excitatory synaptic currents along a dendrite sums bipolar cell inputs at the dendritic tip preferentially for stimulus motion in the centrifugal direction; (2) a "space-time" mechanism that relies on differences in the time-courses of proximal and distal bipolar cell inputs to favor centrifugal stimulus motion. To explore the contributions of these two mechanisms in the primate, we developed a realistic computational model based on connectomic reconstruction of a macaque starburst cell and the distribution of its synaptic inputs from sustained and transient bipolar cell types. Our model suggests that both mechanisms can initiate direction selectivity in starburst dendrites, but their contributions differ depending on the spatiotemporal properties of the stimulus. Specifically, the morphological mechanism dominates when small visual objects are moving at high velocities, and the space-time mechanism contributes most for large visual objects moving at low velocities.
Impact of glaucoma on the spatial frequency processing of scenes in central vision
Glaucoma is an eye disease characterized by a progressive vision loss usually starting in peripheral vision. However, a deficit for scene categorization is observed even in the preserved central vision of patients with glaucoma. We assessed the processing and integration of spatial frequencies in the central vision of patients with glaucoma during scene categorization, considering the severity of the disease, in comparison to age-matched controls. In the first session, participants had to categorize scenes filtered in low-spatial frequencies (LSFs) and high-spatial frequencies (HSFs) as a natural or an artificial scene. Results showed that the processing of spatial frequencies was impaired only for patients with severe glaucoma, in particular for HFS scenes. In the light of proactive models of visual perception, we investigated how LSF could guide the processing of HSF in a second session. We presented hybrid scenes (combining LSF and HSF from two scenes belonging to the same or different semantic category). Participants had to categorize the scene filtered in HSF while ignoring the scene filtered in LSF. Surprisingly, results showed that the semantic influence of LSF on HSF was greater for patients with early glaucoma than controls, and then disappeared for the severe cases. This study shows that a progressive destruction of retinal ganglion cells affects the spatial frequency processing in central vision. This deficit may, however, be compensated by increased reliance on predictive mechanisms at early stages of the disease which would however decline in more severe cases.
Overall patterns of eye-specific retino-geniculo-cortical projections to layers III, IV, and VI in primary visual cortex of the greater galago (), and correlation with cytochrome oxidase blobs
Studies in the greater galago have not provided a comprehensive description of the organization of eye-specific retino-geniculate-cortical projections to the recipient layers in V1. Here we demonstrate the overall patterns of ocular dominance domains in layers III, IV, and VI revealed following a monocular injection of the transneuronal tracer wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP). We also correlate these patterns with the array of cytochrome oxidase (CO) blobs in tangential sections through the unfolded and flattened cortex. In layer IV, we observed for the first time that eye-specific domains form an interconnected pattern of bands 200-250 μm wide arranged such that they do not show orientation bias and do not meet the V1 border at right angles, as is the case in macaques. We also observed distinct WGA-HRP labeled patches in layers III and VI. The patches in layer III, likely corresponding to patches of K lateral geniculate nucleus (LGN) input, align with layer IV ocular dominance columns (ODCs) of the same eye dominance and overlap partially with virtually all CO blobs in both hemispheres, implying that CO blobs receive K LGN input from both eyes. We further found that CO blobs straddle the border between layer IV ODCs, such that the distribution of CO staining is approximately equal over ipsilateral and contralateral ODCs. These results, together with studies showing that a high percentage of cells in CO blobs are monocular, suggest that CO blobs consist of ipsilateral and contralateral subregions that are in register with underlying layer IV ODCs of the same eye dominance. In macaques and humans, CO blobs are centered on ODCs in layer IV. Our finding that CO blobs in galago straddle the border of neighboring layer IV ODCs suggests that this novel feature may represent an alternative way by which visual information is processed by eye-specific modular architecture in mammalian V1.
Differences in chromatic noise suppression of luminance contrast discrimination in young and elderly people
Aging causes impairment of contrast sensitivity and chromatic discrimination, leading to changes in the perceptual interactions between color and luminance information. We aimed to investigate the influence of chromatic noise on luminance contrast thresholds in young and older adults. Forty participants were divided equally into Young (29.6 ± 6.3-year-old) and Elderly Groups (57.8 ± 6.6-year-old). They performed a luminance contrast discrimination task in the presence of chromatic noise maskers using a mosaic stimulus in a mosaic background. Four chromatic noise masking protocols were applied (protan, deutan, tritan, and no-noise protocols). We found that luminance contrast thresholds were significantly elevated by the addition of chromatic noise in both age groups ( < 0.05). In the Elderly group, but not the younger group, thresholds obtained in the tritan protocol were lower than those obtained from protan and deutan protocols ( < 0.05). For all protocols, the luminance contrast thresholds of elderly participants were higher than in young people ( < 0.01). Tritan chromatic noise was less effective in inhibiting luminance discrimination in elderly participants.
Glial cell response to constant low light exposure in rat retina
To study the macroglia and microglia and the immune role in long-time light exposure in rat eyes, we performed glial cell characterization along the time-course of retinal degeneration induced by chronic exposure to low-intensity light. Animals were exposed to light for periods of 2, 4, 6, or 8 days, and the retinal glial response was evaluated by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. Retinal cells presented an increased expression of the macroglia marker GFAP, as well as increased mRNA levels of microglia markers Iba1 and CD68 after 6 days. Also, at this time-point, we found a higher number of Iba1-positive cells in the outer nuclear layer area; moreover, these cells showed the characteristic activated-microglia morphology. The expression levels of immune mediators TNF, IL-6, and chemokines CX3CR1 and CCL2 were also significantly increased after 6 days. All the events of glial activation occurred after 5-6 days of constant light exposure, when the number of photoreceptor cells has already decreased significantly. Herein, we demonstrated that glial and immune activation are secondary to neurodegeneration; in this scenario, our results suggest that photoreceptor death is an early event that occurs independently of glial-derived immune responses.
Classification of pseudocalcium visual responses from mouse retinal ganglion cells-CORRIGENDUM
The mosaic of AII amacrine cell bodies in rat retina is indistinguishable from a random distribution
The vertebrate retina contains a large number of different types of neurons that can be distinguished by their morphological properties. Assuming that no location should be without a contribution from the circuitry and function linked to a specific type of neuron, it is expected that the dendritic trees of neurons belonging to a type will cover the retina in a regular manner. Thus, for most types of neurons, the contribution to visual processing is thought to be independent of the exact location of individual neurons across the retina. Here, we have investigated the distribution of AII amacrine cells in rat retina. The AII is a multifunctional amacrine cell found in mammals and involved in synaptic microcircuits that contribute to visual processing under both scotopic and photopic conditions. Previous investigations have suggested that AIIs are regularly distributed, with a nearest-neighbor distance regularity index of ~4. It has been argued, however, that this presumed regularity results from treating somas as points, without taking into account their actual spatial extent which constrains the location of other cells of the same type. When we simulated random distributions of cell bodies with size and density similar to real AIIs, we confirmed that the simulated distributions could not be distinguished from the distributions observed experimentally for AIIs in different regions and eccentricities of the retina. The developmental mechanisms that generate the observed distributions of AIIs remain to be investigated.
Phosphorylation of cysteine string protein-α up-regulates the frequency of cholinergic waves starburst amacrine cells
During the first postnatal week in rodents, cholinergic retinal waves initiate in starburst amacrine cells (SACs), propagating to retinal ganglion cells (RGCs) and visual centers, essential for visual circuit refinement. By modulating exocytosis in SACs, dynamic changes in the protein kinase A (PKA) activity can regulate the spatiotemporal patterns of cholinergic waves. Previously, cysteine string protein-α (CSPα) is found to interact with the core exocytotic machinery by PKA-mediated phosphorylation at serine 10 (S10). However, whether PKA-mediated CSPα phosphorylation may regulate cholinergic waves via SACs remains unknown. Here, we examined how CSPα phosphorylation in SACs regulates cholinergic waves. First, we identified that CSPα1 is the major isoform in developing rat SACs and the inner plexiform layer during the first postnatal week. Using SAC-specific expression, we found that the CSPα1-PKA-phosphodeficient mutant (CSP-S10A) decreased wave frequency, but did not alter the wave spatial correlation compared to control, wild-type CSPα1 (CSP-WT), or two PKA-phosphomimetic mutants (CSP-S10D and CSP-S10E). These suggest that CSPα-S10 phosphodeficiency in SACs dampens the frequency of cholinergic waves. Moreover, the level of phospho-PKA substrates was significantly reduced in SACs overexpressing CSP-S10A compared to control or CSP-WT, suggesting that the dampened wave frequency is correlated with the decreased PKA activity. Further, compared to control or CSP-WT, CSP-S10A in SACs reduced the periodicity of wave-associated postsynaptic currents (PSCs) in neighboring RGCs, suggesting that these RGCs received the weakened synaptic inputs from SACs overexpressing CSP-S10A. Finally, CSP-S10A in SACs decreased the PSC amplitude and the slope to peak PSC compared to control or CSP-WT, suggesting that CSPα-S10 phosphodeficiency may dampen the speed of the SAC-RGC transmission. Thus, via PKA-mediated phosphorylation, CSPα in SACs may facilitate the SAC-RGC transmission, contributing to the robust frequency of cholinergic waves.
DeBruyn and Casagrande manuscripts on tree shrew retinal ganglion cells as a basis for cross-species retina research
The purpose of this brief communication is to make publicly available three unpublished manuscripts on the organization of retinal ganglion cells in the tree shrew. The manuscripts were authored in 1986 by Dr. Edward DeBruyn, a PhD student in the laboratory of the late Dr. Vivien Casagrande at Vanderbilt University. As diurnal animals closely related to primates, tree shrews are ideally suited for comparative analyses of visual structures including the retina. We hope that providing this basic information in a citable form inspires other groups to pursue further characterization of the tree shrew retina using modern techniques.
Classification of pseudocalcium visual responses from mouse retinal ganglion cells
Recently, a detailed catalog of 32 retinal ganglion cell (RGC) visual response patterns in mouse has emerged. However, the 10,000 samples required for this catalog-based on fluorescent signals from a calcium indicator dye-are much harder to acquire from the extracellular spike train recordings underlying our bionic vision research. Therefore, we sought to convert spike trains into pseudocalcium signals so that our data could be directly matched to the 32 predefined, calcium signal-based groups. A microelectrode array (MEA) was used to record spike trains from mouse RGCs of 29 retinas. Visual stimuli were adapted from the Baden et al. study; including moving bars, full-field contrast and temporal frequency chirps, and black-white and UV-green color flashes. Spike train histograms were converted into pseudocalcium traces with an OGB-1 convolution kernel. Response features were extracted using sparse principal components analysis to match each RGC to one of the 32 RGC groups. These responses mapped onto of the 32 previously described groups; however, some of the groups remained unmatched. Thus, adaptation of the Baden et al. methodology for MEA recordings of spike trains instead of calcium recordings was partially successful. Different classification methods, however, will be needed to define clear RGC groups from MEA data for our bionic vision research. Nevertheless, others may pursue a pseudocalcium approach to reconcile spike trains with calcium signals. This work will help to guide them on the limitations and potential pitfalls of such an approach.
Morphological and electrophysiological specializations of photoreceptors in the love spot of hover fly Volucella pellucens
Studies of functional variability in the compound eyes of flies reveal superior temporal resolution of photoreceptors from the frontal areas that mediate binocular vision, and in males mate recognition and pursuit. However, the mechanisms underlying differences in performance are not known. Here, we investigated properties of hover fly Volucella pellucens photoreceptors from two regions of the retina, the frontal-dorsal "love spot" and the lateral one. Morphologically, the microvilli of the frontal-dorsal photoreceptors were relatively few in number per rhabdomere cross-section, short and narrow. In electrophysiological experiments involving stimulation with prolonged white-noise and natural time intensity series, frontal-dorsal photoreceptors demonstrated comparatively high corner frequencies and information rates. Investigation of possible mechanisms responsible for their superior performance revealed significant differences in the properties of quantum bumps, and, unexpectedly, relatively high absolute sensitivity of the frontal-dorsal photoreceptors. Analysis of light adaptation indicated that photoreceptors from two regions adapt similarly but because frontal-dorsal photoreceptors were depolarized much stronger by the same stimuli than the lateral photoreceptors, they reached a deeper state of adaptation associated with higher corner frequencies of light response. Recordings from the photoreceptor axons were characterized by spike-like events that can significantly expand the frequency response range. Seamless integration of spikes into the graded voltage responses was enabled by light adaptation mechanisms that accelerate kinetics and decrease duration of depolarizing light response transients.
Protein kinase A regulation of pigment granule motility in retinal pigment epithelial cells from fish, spp
Retinomotor movements include elongation and contraction of rod and cone photoreceptors, and mass migration of melanin-containing pigment granules (melanosomes) of the retinal pigment epithelium (RPE) within the eyes of fish, frogs, and other lower vertebrates. Eyes of these animals do not contain dilatable pupils; therefore the repositioning of the rods and cones and a moveable curtain of pigment granules serve to modulate light intensity within the eye. RPE from sunfish (Lepomis spp.) can be isolated from the eye and dissociated into single cells, allowing in vitro studies of the cytoskeletal and regulatory mechanisms of organelle movement. Pigment granule aggregation from distal tips of apical projections into the cell body can be triggered by the application of underivatized cAMP, and dispersion is effected by cAMP washout in the presence of dopamine. While the phenomenon of cAMP-dependent pigment granule aggregation in isolated RPE was described many years ago, whether cAMP acts through the canonical cAMP-PKA pathway to stimulate motility has never been demonstrated. Here, we show that pharmacological inhibition of PKA blocks pigment granule aggregation, and microinjection of protein kinase A catalytic subunit triggers pigment granule aggregation. Treatment with a cAMP agonist that activates the Rap GEF, Epac (Effector protein activated by cAMP), had no effect on pigment granule position. Taken together, these results confirm that cAMP activates RPE pigment granule motility by the canonical cAMP-PKA pathway. Isolated RPE cells labeled with antibodies against PKA RIIα and against PKA-phosphorylated serine/threonine amino acids show diffuse, punctate labeling throughout the RPE cell body and apical projections. Immunoblotting of RPE lysates using the anti-PKA substrate antibody demonstrated seven prominent bands; two bands in particular at 27 and 64 kD showed increased levels of phosphorylation in the presence of cAMP, indicating their phosphorylation could contribute to the pigment granule aggregation mechanism.
Structure and function of the gap junctional network of photoreceptive ganglion cells
Intrinsically photosensitive retinal ganglion cells (ipRGCs) signal not only anterogradely to drive behavioral responses, but also retrogradely to some amacrine interneurons to modulate retinal physiology. We previously found that all displaced amacrine cells with spiking, tonic excitatory photoresponses receive gap-junction input from ipRGCs, but the connectivity patterns and functional roles of ipRGC-amacrine coupling remained largely unknown. Here, we injected PoPro1 fluorescent tracer into all six types of mouse ipRGCs to identify coupled amacrine cells, and analyzed the latter's morphological and electrophysiological properties. We also examined how genetically disrupting ipRGC-amacrine coupling affected ipRGC photoresponses. Results showed that ipRGCs couple with not just ON- and ON/OFF-stratified amacrine cells in the ganglion-cell layer as previously reported, but also OFF-stratified amacrine cells in both ganglion-cell and inner nuclear layers. M1- and M3-type ipRGCs couple mainly with ON/OFF-stratified amacrine cells, whereas the other ipRGC types couple almost exclusively with ON-stratified ones. ipRGCs transmit melanopsin-based light responses to at least 93% of the coupled amacrine cells. Some of the ON-stratifying ipRGC-coupled amacrine cells exhibit transient hyperpolarizing light responses. We detected bidirectional electrical transmission between an ipRGC and a coupled amacrine cell, although transmission was asymmetric for this particular cell pair, favoring the ipRGC-to-amacrine direction. We also observed electrical transmission between two amacrine cells coupled to the same ipRGC. In both scenarios of coupling, the coupled cells often spiked synchronously. While ipRGC-amacrine coupling somewhat reduces the peak firing rates of ipRGCs' intrinsic melanopsin-based photoresponses, it renders these responses more sustained and longer-lasting. In summary, ipRGCs' gap junctional network involves more amacrine cell types and plays more roles than previously appreciated.
Structural and white matter changes associated with duration of Braille education in early and late blind children
In early (EB) and late blind (LB) children, vision deprivation produces cross-modal plasticity in the visual cortex. The progression of structural- and tract-based spatial statistics changes in the visual cortex in EB and LB, as well as their impact on global cognition, have yet to be investigated. The purpose of this study was to determine the cortical thickness (CT), gyrification index (GI), and white matter (WM) integrity in EB and LB children, as well as their association to the duration of blindness and education. Structural and diffusion tensor imaging data were acquired in a 3T magnetic resonance imaging in EB and LB children (n = 40 each) and 30 sighted controls (SCs) and processed using CAT12 toolbox and FSL software. Two sample t-test was used for group analyses with P < 0.05 (false discovery rate-corrected). Increased CT in visual, sensory-motor, and auditory areas, and GI in bilateral visual cortex was observed in EB children. In LB children, the right visual cortex, anterior-cingulate, sensorimotor, and auditory areas showed increased GI. Structural- and tract-based spatial statistics changes were observed in anterior visual pathway, thalamo-cortical, and corticospinal tracts, and were correlated with education onset and global cognition in EB children. Reduced impairment in WM, increased CT and GI and its correlation with global cognitive functions in visually impaired children suggests cross-modal plasticity due to adaptive compensatory mechanism (as compared to SCs). Reduced CT and increased FA in thalamo-cortical areas in EB suggest synaptic pruning and alteration in WM integrity. In the visual cortical pathway, higher education and the development of blindness modify the morphology of brain areas and influence the probabilistic tractography in EB rather than LB.
Blockade of retinal or cortical activity does not prevent the development of callosal patches normally associated with ocular dominance columns in primary visual cortex
Callosal patches in primary visual cortex of Long Evans rats, normally associated with ocular dominance columns, emerge by postnatal day 10 (P10), but they do not form in rats monocularly enucleated a few days before P10. We investigated whether we could replicate the results of monocular enucleation by using tetrodotoxin (TTX) to block neural activity in one eye, or in primary visual cortex. Animals received daily intravitreal (P6-P9) or intracortical (P7-P9) injections of TTX, and our physiological evaluation of the efficacy of these injections indicated that the blockade induced by a single injection lasted at least 24 h. Four weeks later, the patterns of callosal connections in one hemisphere were revealed after multiple injections of horseradish peroxidase in the other hemisphere. We found that in rats receiving either intravitreal or cortical injections of TTX, the patterns of callosal patches analyzed in tangential sections from the flattened cortex were not significantly different from the pattern in normal rats. Our findings, therefore, suggest that the effects of monocular enucleation on the distribution of callosal connections are not due to the resulting imbalance of afferent ganglion cell activity, and that factors other than neural activity are likely involved.