Stress induced aging in mouse eye.

Aging, a universal process that affects all cells in an organism, is a major risk factor for a group of neuropathies called glaucoma, where elevated intraocular pressure is one of the known stresses affecting the tissue. Our understanding of molecular impact of aging on response to stress in retina is very limited; therefore, we developed a new mouse model to approach this question experimentally. Here we show that susceptibility to response to stress increases with age and is primed on chromatin level. We demonstrate that ocular hypertension activates a stress response that is similar to natural aging and involves activation of inflammation and senescence. We show that multiple instances of pressure elevation cause aging of young retina as measured on transcriptional and DNA methylation level and are accompanied by local histone modification changes. Our data show that repeated stress accelerates appearance of aging features in tissues and suggest chromatin modifications as the key molecular components of aging. Lastly, our work further emphasizes the importance of early diagnosis and prevention as well as age-specific management of age-related diseases, including glaucoma.

Visual System Hyperexcitability and Compromised V1 Receptive Field Properties in Early-Stage Retinitis Pigmentosa in Mice.

Inherited retinal degenerative diseases are a prominent cause of blindness. Although mutations causing death of photoreceptors are mostly known, the pathophysiology downstream in the inner retina and along the visual pathway is incompletely characterized in the earliest disease stages. Here, we investigated retinal, midbrain and cortical visual function using electroretinography (ERG), the optomotor response (OMR), visual evoked potentials (VEPs), respectively, and single unit electrophysiology at the primary visual cortex (V1) in light-adapted juvenile (approximately one-month-old) and young adult (three-month-old) RhoP23H/WT mice, representative of early-stage retinitis pigmentosa (RP). Photopic ERG revealed up to ∼30% hypersensitivity to light in RhoP23H/WT mice, as measured by the light intensity required to generate half-maximal b-wave (I50 parameter). RhoP23H/WT mice also showed increased OMRs toward low spatial frequency (SF) drifting gratings, indicative of visual overexcitation at the midbrain level. At the V1 level, VEPs and single-cell recordings revealed prominent hyperexcitability in the juvenile RhoP23H/WT mice. Mean VEP amplitudes for light ON stimuli were nearly doubled in one-month-old RhoP23H/WT mice compared with controls, and more than doubled for light OFF. Single-cell recordings showed a significantly increased spontaneous V1 neuron firing in the RhoP23H/WT mice, and persistent contrast and temporal sensitivities. In contrast, direction selectivity was severely compromised. Our data suggest that during early RP, the visual pathway becomes hyperexcited. This could have both compensatory and deleterious consequences for visual behavior. Further studies on the mechanisms of hyperexcitability are warranted as this could lead to therapeutic interventions for RP.

Brain-wide reconstruction of inhibitory circuits after traumatic brain injury.

Despite the fundamental importance of understanding the brain's wiring diagram, our knowledge of how neuronal connectivity is rewired by traumatic brain injury remains remarkably incomplete. Here we use cellular resolution whole-brain imaging to generate brain-wide maps of the input to inhibitory neurons in a mouse model of traumatic brain injury. We find that somatostatin interneurons are converted into hyperconnected hubs in multiple brain regions, with rich local network connections but diminished long-range inputs, even at areas not directly damaged. The loss of long-range input does not correlate with cell loss in distant brain regions. Interneurons transplanted into the injury site receive orthotopic local and long-range input, suggesting the machinery for establishing distant connections remains intact even after a severe injury. Our results uncover a potential strategy to sustain and optimize inhibition after traumatic brain injury that involves spatial reorganization of the direct inputs to inhibitory neurons across the brain.

In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration.

Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.

Inhibition of ceramide accumulation in AdipoR1-/- mice increases photoreceptor survival and improves vision.

Adiponectin receptor 1 (ADIPOR1) is a lipid and glucose metabolism regulator that possesses intrinsic ceramidase activity. Mutations of the ADIPOR1 gene have been associated with nonsyndromic and syndromic retinitis pigmentosa. Here, we show that the absence of AdipoR1 in mice leads to progressive photoreceptor degeneration, significant reduction of electroretinogram amplitudes, decreased retinoid content in the retina, and reduced cone opsin expression. Single-cell RNA-Seq results indicate that ADIPOR1 encoded the most abundantly expressed ceramidase in mice and one of the 2 most highly expressed ceramidases in the human retina, next to acid ceramidase ASAH1. We discovered an accumulation of ceramides in the AdipoR1-/- retina, likely due to insufficient ceramidase activity for healthy retina function, resulting in photoreceptor death. Combined treatment with desipramine/L-cycloserine (DC) lowered ceramide levels and exerted a protective effect on photoreceptors in AdipoR1-/- mice. Moreover, we observed improvement in cone-mediated retinal function in the DC-treated animals. Lastly, we found that prolonged DC treatment corrected the electrical responses of the primary visual cortex to visual stimuli, approaching near-normal levels for some parameters. These results highlight the importance of ADIPOR1 ceramidase in the retina and show that pharmacological inhibition of ceramide generation can provide a therapeutic strategy for ADIPOR1-related retinopathy.

Traumatic brain injury to primary visual cortex produces long-lasting circuit dysfunction.

Primary sensory areas of the mammalian neocortex have a remarkable degree of plasticity, allowing neural circuits to adapt to dynamic environments. However, little is known about the effects of traumatic brain injury on visual circuit function. Here we used anatomy and in vivo electrophysiological recordings in adult mice to quantify neuron responses to visual stimuli two weeks and three months after mild controlled cortical impact injury to primary visual cortex (V1). We found that, although V1 remained largely intact in brain-injured mice, there was ~35% reduction in the number of neurons that affected inhibitory cells more broadly than excitatory neurons. V1 neurons showed dramatically reduced activity, impaired responses to visual stimuli and weaker size selectivity and orientation tuning in vivo. Our results show a single, mild contusion injury produces profound and long-lasting impairments in the way V1 neurons encode visual input. These findings provide initial insight into cortical circuit dysfunction following central visual system neurotrauma.

Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.

Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of a lentivirus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.

Projections between visual cortex and pulvinar in the rat.

The extrageniculate visual pathway, which carries visual information from the retina through the superficial layers of the superior colliculus and the pulvinar, is poorly understood. The pulvinar is thought to modulate information flow between cortical areas, and has been implicated in cognitive tasks like directing visually guided actions. In order to better understand the underlying circuitry, we performed retrograde injections of modified rabies virus in the visual cortex and pulvinar of the Long-Evans rat. We found a relatively small population of cells projecting to primary visual cortex (V1), compared to a much larger population projecting to higher visual cortex. Reciprocal corticothalamic projections showed a similar result, implying that pulvinar does not play as big a role in directly modulating rodent V1 activity as previously thought.

Publisher Correction: Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Visual Response Characteristics in Lateral and Medial Subdivisions of the Rat Pulvinar.

The pulvinar is a higher-order thalamic relay and a central component of the extrageniculate visual pathway, with input from the superior colliculus and visual cortex and output to all of visual cortex. Rodent pulvinar, more commonly called the lateral posterior nucleus (LP), consists of three highly-conserved subdivisions, and offers the advantage of simplicity in its study compared to more subdivided primate pulvinar. Little is known about receptive field properties of LP, let alone whether functional differences exist between different LP subdivisions, making it difficult to understand what visual information is relayed and what kinds of computations the pulvinar might support. Here, we characterized single-cell response properties in two V1 recipient subdivisions of rat pulvinar, the rostromedial (LPrm) and lateral (LPl), and found that a fourth of the cells were selective for orientation, compared to half in V1, and that LP tuning widths were significantly broader. Response latencies were also significantly longer and preferred size more than three times larger on average than in V1; the latter suggesting pulvinar as a source of spatial context to V1. Between subdivisons, LPl cells preferred higher temporal frequencies, whereas LPrm showed a greater degree of direction selectivity and pattern motion detection. Taken together with known differences in connectivity patterns, these results suggest two separate visual feature processing channels in the pulvinar, one in LPl related to higher speed processing which likely derives from superior colliculus input, and the other in LPrm for motion processing derived through input from visual cortex. SIGNIFICANCE STATEMENT: The pulvinar has a perplexing role in visual cognition as no clear link has been found between the functional properties of its neurons and behavioral deficits that arise when it is damaged. The pulvinar, called the lateral posterior nucleus (LP) in rats, is a higher order thalamic relay with input from the superior colliculus and visual cortex and output to all of visual cortex. By characterizing single-cell response properties in anatomically distinct subdivisions we found two separate visual feature processing channels in the pulvinar, one in lateral LP related to higher speed processing which likely derives from superior colliculus input, and the other in rostromedial LP for motion processing derived through input from visual cortex.

Cell type specific tracing of the subcortical input to primary visual cortex from the basal forebrain.

The basal forebrain provides cholinergic inputs to primary visual cortex (V1) that play a key modulatory role on visual function. While basal forebrain afferents terminate in the infragranular layers of V1, acetylcholine is delivered to more superficial layers through volume transmission. Nevertheless, direct synaptic contact in deep layers 5 and 6 may provide a more immediate effect on V1 modulation. Using helper viruses with cell type specific promoters to target retrograde infection of pseudotyped and genetically modified rabies virus evidence was found for direct synaptic input onto V1 inhibitory neurons. These inputs were similar in number to geniculocortical inputs and, therefore, considered robust. In contrast, while clear evidence for dorsal lateral geniculate nucleus input to V1 excitatory neurons was found, there was no evidence of direct synaptic input from the basal forebrain. These results suggest a direct and more immediate influence of the basal forebrain on local V1 inhibition.

Detailed Visual Cortical Responses Generated by Retinal Sheet Transplants in Rats with Severe Retinal Degeneration.

To combat retinal degeneration, healthy fetal retinal sheets have been successfully transplanted into both rodent models and humans, with synaptic connectivity between transplant and degenerated host retina having been confirmed. In rodent studies, transplants have been shown to restore responses to flashes of light in a region of the superior colliculus corresponding to the location of the transplant in the host retina. To determine the quality and detail of visual information provided by the transplant, visual responsivity was studied here at the level of visual cortex where higher visual perception is processed. For our model, we used the transgenic Rho-S334ter line-3 rat (both sexes), which loses photoreceptors at an early age and is effectively blind at postnatal day 30. These rats received fetal retinal sheet transplants in one eye between 24 and 40 d of age. Three to 10 months following surgery, visually responsive neurons were found in regions of primary visual cortex matching the transplanted region of the retina that were as highly selective as normal rat to stimulus orientation, size, contrast, and spatial and temporal frequencies. Conversely, we found that selective response properties were largely absent in nontransplanted line-3 rats. Our data show that fetal retinal sheet transplants can result in remarkably normal visual function in visual cortex of rats with a degenerated host retina and represents a critical step toward developing an effective remedy for the visually impaired human population.SIGNIFICANCE STATEMENT Age-related macular degeneration and retinitis pigmentosa lead to profound vision loss in millions of people worldwide. Many patients lose both retinal pigment epithelium and photoreceptors. Hence, there is a great demand for the development of efficient techniques that allow for long-term vision restoration. In this study, we transplanted dissected fetal retinal sheets, which can differentiate into photoreceptors and integrate with the host retina of rats with severe retinal degeneration. Remarkably, we show that transplants generated visual responses in cortex similar in quality to normal rats. Furthermore, transplants preserved connectivity within visual cortex and the retinal relay from the lateral geniculate nucleus to visual cortex, supporting their potential application in curing vision loss associated with retinal degeneration.

Differences in orientation tuning between pinwheel and domain neurons in primary visual cortex depend on contrast and size.

Intrinsic signal optical imaging reveals a highly modular map of orientation preference in the primary visual cortex (V1) of several species. This orientation map is characterized by domains and pinwheels where local circuitry is either more or less orientation selective, respectively. It has now been repeatedly demonstrated that neurons in pinwheels tend to be more broadly tuned to orientation, likely due to the broad range of orientation preference of the neighboring neurons forming pinwheels. However, certain stimulus conditions, such as a decrease in contrast or an increase in size, significantly sharpen tuning widths of V1 neurons. Here, we find that pinwheel neuron tuning widths are broader than domain neurons only for high contrast, optimally sized stimuli, conditions that maximize excitation through feedforward, and local cortical processing. When contrast was lowered or size increased, orientation tuning width sharpened and became equal. These latter conditions are conducive to less local excitation either through lower feedforward drive or by surround suppression arising from long-range cortical circuits. Tuning width differences between pinwheel and domain neurons likely arise through more local circuitry and are overcome through recruitment of longer-range cortical circuits.

V1 connections reveal a series of elongated higher visual areas in the California ground squirrel, Otospermophilus beecheyi.

For studies of visual cortex organization, mouse is becoming an increasingly more often used model. In addition to its genetic tractability, the relatively small area of cortical surface devoted to visual processing simplifies efforts in relating the structure of visual cortex to visual function. However, the nature of this compact organization can make some comparisons to the much larger non-human primate visual cortex difficult. The squirrel, as a highly visual rodent offers a useful means for better understanding how mouse and monkey cortical organization compares. More in line with primates than their nocturnal rodent cousin, squirrels rely much more on sight and have evolved a larger expanse of cortex devoted to visual processing. To reveal the detailed organization of visual cortex in squirrels, we injected a highly sensitive monosynaptic retrograde tracer (glycoprotein deleted rabies virus) into several locations of primary visual cortex (V1) in California ground squirrels. The resulting pattern of connectivity revealed an organizational scheme in the squirrel that retains some of the basic features of the mouse visual cortex along the medial and posterior borders of V1, but unlike mouse has an elaborate and extensive pattern laterally that is more similar to the early visual cortex organization found in monkeys. In this way, we show that the squirrel can serve as a useful model for comparison to both mouse and primate visual systems, and may help facilitate comparisons between these two very different yet widely used animal models of visual processing.

Resolving the organization of the territory of the third visual area: a new proposal.

In primates, the cortex adjoining the rostral border of V2 has been variously interpreted as belonging to a single visual area, V3, with dorsal V3 (V3d) representing the lower visual quadrant and ventral V3 (V3v) representing the upper visual quadrant, V3d and V3v constituting separate, incomplete visual areas, V3d and ventral posterior (VP), or V3d being divided into several visual areas, including a dorsomedial (DM) visual area, a medial visual area (M), and dorsal extension of VP (or VLP). In our view, the evidence from V1 connections strongly supports the contention that V3v and V3d are parts of a single visual area, V3, and that DM is a separate visual area along the rostral border of V3d. In addition, the retinotopy revealed by V1 connection patterns, microelectrode mapping, optical imaging mapping, and functional magnetic resonance imaging (fmri) mapping indicates that much of the proposed territory of V3d corresponds to V3. Yet, other evidence from microelectrode mapping and anatomical connection patterns supports the possibility of an upper quadrant representation along the rostral border of the middle of dorsal V2 (V2d), interpreted as part of DM or DM plus DI, and along the midline end of V2d, interpreted as the visual area M. While the data supporting these different interpretations appear contradictory, they also seem, to some extent, valid. We suggest that V3d may have a gap in its middle, possibly representing part of the upper visual quadrant that is not part of DM. In addition, another visual area, M, is likely located at the DM tip of V3d. There is no evidence for a similar disruption of V3v. For the present, we favor continuing the traditional concept of V3 with the possible modification of a gap in V3d in at least some primates.

Contrast invariance of orientation tuning in cat primary visual cortex neurons depends on stimulus size.

KEY POINTS: The process of orientation tuning is an important and well-characterized feature of neurons in primary visual cortex. The combination of ascending and descending circuits involved is not only relevant to understanding visual processing but the function of neocortex in general. The classic feed-forward model of orientation tuning predicts a broadening effect due to increasing contrast; yet, experimental results consistently report contrast invariance. We show here that contrast invariance actually depends on stimulus size such that large stimuli extending beyond the neuron's receptive field engage circuits that promote invariance, whereas optimally sized, smaller stimuli result in contrast variance that is more in line with the classical orientation tuning model. These results illustrate the importance of optimizing stimulus parameters to best reflect the sensory pathways under study and provide new clues about different circuits that may be involved in variant and invariant response properties. ABSTRACT: Selective response to stimulus orientation is a key feature of neurons in primary visual cortex, yet the underlying mechanisms generating orientation tuning are not fully understood. The combination of feed-forward and cortical mechanisms involved is not only relevant to understanding visual processing but the function of neocortex in general. The classic feed-forward model predicts that orientation tuning should broaden considerably with increasing contrast; however, experimental results consistently report contrast invariance. We show here, in primary visual cortex of anaesthetized cats under neuromuscular blockade, that contrast invariance occurs when visual stimuli are large enough to include the extraclassical surround (ECS), which is likely to involve circuits of suppression that may not be entirely feed-forward in origin. On the other hand, when stimulus size is optimized to the classical receptive field of each neuron, the population average shows a statistically significant 40% increase in tuning width at high contrast, demonstrating that contrast variance of orientation tuning can occur. Conversely, our results also suggest that the phenomenon of contrast invariance relies in part on the presence of the ECS. Moreover, these results illustrate the importance of optimizing stimulus parameters to best reflect the neural pathways under study.

Distribution of cortical neurons projecting to the superior colliculus in macaque monkeys.

To better reveal the pattern of corticotectal projections to the superficial layers of the superior colliculus (SC), we made a total of ten retrograde tracer injections into the SC of three macaque monkeys (Macaca mulatta). The majority of these injections were in the superficial layers of the SC, which process visual information. To isolate inputs to the purely visual layers in the superficial SC from those inputs to the motor and multisensory layers deeper in the SC, two injections were placed to include the intermediate and deep layers of the SC. In another case, an injection was placed in the medial pulvinar, a nucleus not known to be strongly connected with visual cortex, to identify possible projections from tracer spread past the lateral boundary of the SC. Four conclusions are supported by the results: 1) all early visual areas of cortex, including V1, V2, V3, and the middle temporal area, project to the superficial layers of the SC; 2) with the possible exception of the frontal eye field, few areas of cortex outside of the early visual areas project to the superficial SC, although many do, however, project to the intermediate and deep layers of the SC; 3) roughly matching retinotopy is conserved in the projections of visual areas to the SC; and 4) the projections from different visual areas are similarly dense, although projections from early visual areas appear somewhat denser than those of higher order visual areas in macaque cortex.

Very-long-range disynaptic V1 connections through layer 6 pyramidal neurons revealed by transneuronal tracing with rabies virus.

Neurons in primary visual cortex (V1) integrate across the representation of the visual field through networks of long-range projecting pyramidal neurons. These projections, which originate from within V1 and through feedback from higher visual areas, are likely to play a key role in such visual processes as low contrast facilitation and extraclassical surround suppression. The extent of the visual field representation covered by feedback is generally much larger than that covered through monosynaptic horizontal connections within V1, and, although it may be possible that multisynaptic horizontal connections across V1 could also lead to more widespread spatial integration, nothing is known regarding such circuits. In this study, we used injections of the CVS-11 strain of rabies virus to examine disynaptic long-range horizontal connections within macaque monkey V1. Injections were made around the representation of 5° eccentricity in the lower visual field. Along the opercular surface of V1, we found that the majority of connected neurons extended up to 8 mm in most layers, consistent with twice the typically reported distances of monosynaptic connections. In addition, mainly in layer 6, a steady presence of connected neurons within V1 was observed up to 16 mm away. A relatively high percentage of these connected neurons had large-diameter somata characteristic of Meynert cells, which are known to project as far as 8 mm individually. Several neurons, predominantly in layer 6, were also found deep within the calcarine sulcus, reaching as far as 20° of eccentricity, based on estimates, and extending well into the upper visual field representation. Thus, our anatomical results provide evidence for a wide-ranging disynaptic circuit within V1, mediated largely through layer 6, that accounts for integration across a large region of the visual field.

Tracing inputs to inhibitory or excitatory neurons of mouse and cat visual cortex with a targeted rabies virus.

BACKGROUND: Cortical inhibition plays a critical role in controlling and modulating cortical excitation, and a more detailed understanding of the neuronal circuits contributing to each will provide more insight into their roles in complex cortical computations. Traditional neuronal tracers lack a means for easily distinguishing between circuits of inhibitory and excitatory neurons. To overcome this limitation, we have developed a technique for retrogradely labeling inputs to local clusters of inhibitory or excitatory neurons, but not both, using neurotropic adenoassociated and lentiviral vectors, cell-type-specific promoters, and a modified rabies virus. RESULTS: Applied to primary visual cortex (V1) in mouse, the cell-type-specific tracing technique labeled thousands of presynaptically connected neurons and revealed that the dominant source of input to inhibitory and excitatory neurons is local in origin. Neurons in other visual areas are also labeled; the percentage of these intercortical inputs to excitatory neurons is somewhat higher (~20%) than to inhibitory neurons (<10%), suggesting that intercortical connections have less direct control over inhibition. The inputs to inhibitory neurons were also traced in cat V1, and when aligned with the orientation preference map revealed for the first time that long-range inputs to inhibitory neurons are well tuned to orientation. CONCLUSIONS: These novel findings for inhibitory and excitatory circuits in the visual cortex demonstrate the efficacy of our new technique and its ability to work across species, including larger-brained mammals such as the cat. This paves the way for a better understanding of the roles of specific cell types in higher-order perceptual and cognitive processes.

Lack of robust LGN label following transneuronal rabies virus injections into macaque area V4.

In primates, retinal inputs are relayed through the magno- and parvocells of the lateral geniculate nucleus (LGN) indirectly to extrastriate visual cortex. The most direct pathway identified to the extrastriate cortex is a disynaptic one that provides robust magno- and parvocellular inputs to the middle temporal area (MT). The inclusion of parvocells in this projection is somewhat surprising because of their importance for color and form vision, whereas MT is more strictly tuned to velocity. This raises the question of whether areas more involved in color and form processing, such as V4, receive similar projections. We report here on experiments that use rabies virus injections into V4 to retrogradely label mono- and disynaptic inputs. We find only a small number of labeled neurons in the LGN in a pattern consistent with monosynaptic labeling of koniocells, rather than disynaptic labeling of magno- and parvocells. The lack of robust magno- and parvocellular label was not due to ineffective viral transport because in the same cases we find hundreds of neurons labeled in the thalamic reticular nucleus, a structure that can only be labeled disynaptically from the cortex. We also find a complete absence of neurons labeled in V1, but thousands in adjacent areas V2 and V3. This result helps explain the absence of labeled magno- and parvocells in the LGN because disynaptic transport from an extrastriate visual area should require a relay through V1. Taken together, these results suggest that ascending magno/parvocellular inputs to V4 are more hierarchically organized than the relatively direct inputs to MT.