Institut de la Vision

The ascending sensory projections from the dorsal thalamus to the pallium are one of the most distinctive features of the amniote brain. In mammals, these projections can be broadly classified into two main types: (1) those arising from "core" neurons, mostly located in the central region of sensory nuclei, which relay topographic inputs to the intermediate layers of primary sensory cortices, forming a reciprocal circuit with the reticular thalamic nucleus (RTN); and (2) those originating from "matrix" neurons, located in the periphery of sensory nuclei, which project to superficial layers of primary and higher order cortices. To date, both the reciprocal core-RTN circuitry and the matrix projections have only been described in mammals. In this study, we re-examined the organization of thalamic projections to the visual dorsal ventricular ridge (DVR) in pigeons using neural tracing techniques. In addition to the well-known projection from the thalamic nucleus rotundus (Rt) to the ventral layer of the visual DVR (entopallium), we described a reciprocal projection pattern between the Rt and the RTN. Moreover, we identified a second ascending thalamopallial projection, arising from neurons surrounding the Rt and terminating primarily in the intermediate layer of the visual DVR. These neurons form a continuous "matrix" of cells within the dorsal thalamus, which as a group also give rise to axons targeting several associative pallial areas. Furthermore, we found that this "matrix" region receives descending inputs from the motor arcopallium. Collectively, these findings indicate that the avian thalamopallial system is more complex than previously recognized and thus more comparable to its mammalian counterpart.

Brightness and contrast are fundamental features of vision, crucial for object detection, environmental navigation, and feeding. Here, we identify a brightness- and contrast-processing circuit in the zebrafish visual system and uncover the role of leucine-rich repeat neuronal (Lrrn) cell adhesion molecules (CAMs) in regulating its assembly. We show that deep-projecting retinal ganglion cells serve as the first synaptic relay to the brain, requiring Lrrn2 and Lrrn3a for precise axonal targeting within the optic tectum. Using a new reporter line, we achieved high-resolution mapping of this previously undercharacterized vertebrate visual circuit. Genetic disruption of Lrrn CAMs leads to disorganization of the circuit and impairments in brightness and contrast sensitivity, resulting in deficits in visually guided behavior. Additionally, ultrastructural circuit reconstruction and functional imaging analysis identified both its synaptic partners and revealed its critical role in luminance processing. These studies define a core visual processing pathway and establish Lrrn CAMs as essential molecular drivers of its assembly.

ABSTRACT Conjunctival in vitro models present a valuable system to investigate conjunctival tissue homeostasis and pathologies. Combinations of collagen and fibroblasts as a stroma equivalent and the supplementation with serum have been reported to promote the differentiation of epithelial cells. However, how the individual factors affect differentiation of ocular surface cells is insufficiently understood. In this study, we analyzed the effect of serum concentration, a collagen matrix, and fibroblasts on conjunctival differentiation in a 3D in vitro model. For this purpose, we developed a computational analysis pipeline for the quantification of optical coherence tomography (OCT) data sets, allowing a time resolved, non-invasive assessment of conjunctiva epithelium differentiation, including goblet cell density. High-resolution dynamic full-field OCT (D-FFOCT) was employed to verify the identity of goblet cells. Conjunctival markers were further analyzed via histology, real-time quantitative PCR, and ELISA to confirm the data of the OCT analysis pipeline. We found that serum is required to induce epithelial differentiation while higher concentrations of 5 – 10% impaired epithelial development. The culture on a collagen matrix increased conjunctival markers upon stimulation with serum, while the co-culture with fibroblasts increased epithelial stratification. Increased serum concentration resulted in the increased occurrence of goblet cells of up to 20 cells/mm². Altogether, the complementary analyses confirmed the quantified OCT data. Summarized, we identified the combination of serum (3%), collagen, and fibroblasts as a condition resulting in the highest physiological resemblance. Altogether, our study emphasizes the need for fine-tuning of culture conditions for 3D in vitro models. SIGNIFICANCE STATEMENT Physiologically relevant in vitro conjunctiva models are essential for studying ocular surface homeostasis and disease. Our study refines 3D conjunctival culture conditions to more closely resemble native tissue by systematically identifying serum concentration, collagen scaffolds and fibroblasts as key drivers of epithelial differentiation. By applying non-invasive optical coherence tomography analysis, we enable longitudinal assessment of tissue maturation, addressing the need for non-destructive, repeatable tissue analysis. GRAPHICAL ABSTRACT

Perceptual and sensorimotor events are often experienced as temporal patterns, that is, identified as sequences based on their temporal features. While current timing models propose separate mechanisms supporting the processing of single intervals and temporal patterns, they leave partially unclear whether the latter entails the processing of both individual intervals and the overall structure of a pattern, or only one of these features. Here, we narrowed this question down by investigating how violations of regularity within the individual intervals of a temporal sequence (i.e., local violations) and in its overall structure (i.e., global violations) differentially affect its reproduction. We tested these violation effects in three experiments in which the sequences were experienced either in the visual or auditory domain and had either simple or complex structures. Results showed that the precision in reproducing simple visual and auditory patterns was primarily affected by local violations, whereas global violations mostly impacted the reproduction of visual patterns with complex structures. These detrimental effects were partially explained by rescaling and bias effects in the reproduced patterns. Overall, our findings indicate that the processing and reproduction of temporal patterns differentially weigh individual intervals and global structure, depending on sensory modality and, for visual patterns, on structural complexity.

The lateral geniculate nucleus (LGN) of the thalamus is a key link between the retina and visual cortex but our understanding of the properties of neurons in the human LGN is based on recordings in animal models. Here we recorded spiking activity of cells in the LGN of two patients who had electrodes implanted in the LGN as part of their treatment for epilepsy. Human LGN cells responded to strong visual stimulation with high-frequency bursts of spikes. The cells had receptive-field properties resembling those of monkeys with circular ON-OFF sub-fields, red-green opponency in the dorsal layers and preferences for high temporal frequencies in the ventral layers. Responses were largely monocular and the closure of one eye decreased the spontaneous activity of broad-spiking neurons preferring this eye while increasing the activity of neurons with narrower spikes, suggesting that interneurons might gate LGN activity during eye closure.

In classic disease models, removing a pathological insult restores homeostasis. Yet, addiction persists far beyond the period of active drug use. Cocaine abstinence induces changes in gene expression and neuronal signaling in reward-related brain regions that limit recovery during abstinence. We found that 2 weeks of abstinence increased Cartpt (cocaine- and amphetamine-regulated transcript) in the mouse nucleus accumbens and decreased repressive H3K27me3 at the Cartpt locus. While endogenous CART peptide is best described for its anorexigenic function, it is also implicated in human addiction and dopamine homeostasis. To test the causal relevance of Cartpt chromatin remodeling, we used CRISPR-based epigenetic editing tools, dCas9-FOG1 and dCas9-JMJC-ZF, to manipulate H3K27me3 at Cartpt in vivo. Enriching H3K27me3 in D1 neurons repressed Cartpt expression and augmented acquisition and extinction of cocaine preference. These results show that CRISPR epigenetic editing can recapitulate endogenous chromatin states to modulate addiction-related behavior, highlighting broad therapeutic potential of both Cartpt and epigenetic editing.

Human retinal organoids are in vitro 3D structures that recapitulate key molecular and structural characteristics of the in vivo retina. They include all essential retinal cell types including photoreceptors, making them relevant models for preclinical development of gene therapies. A critical knowledge gap exists in understanding their utility for gene editing therapy optimization. We assessed the potential of retinal organoids for optimizing CRISPR-Cas9-mediated gene editing, focusing on the therapeutically relevant RHO gene implicated in autosomal dominant retinitis pigmentosa (adRP). Using retinal organoids, in vitro HEK293T cells, and two humanized mouse models carrying different RHO mutations, we compared editing efficiencies. We observed that retinal organoids have lower transfection efficiency compared to HEK293T cells. Notably, they exhibited editing efficiencies more closely aligned with those found in vivo. We also observed similar delivery patterns of CRISPR-Cas9 tools in both retinal organoids and mouse retinas. These delivery patterns and editing efficiencies remained consistent across dual adeno-associated virus (AAV) systems and transiently delivered ribonucleoprotein complexes. Our findings demonstrate that retinal organoids achieve editing outcomes comparable to those observed in vivo underscoring their utility as part of a preclinical testing platform for genome editing, with implications for advancing gene therapy research in inherited retinal diseases.