Journal of Comparative Neurology

All insects studied to date possess a centrally located group of neuropils, known collectively as the central complex, that has been implicated in sensory integration and motor action selection. Among the functions prescribed to the central complex, none is perhaps as intriguing as its role in orientation and navigation. Neurobiological correlates of both current and desired headings have been described in insect CXs. Despite the diversity of arthropods, understanding of the CX as a navigational center originates entirely from terrestrial insects. Stomatopod crustaceans, commonly referred to as mantis shrimps, form an order of predatory marine crustaceans with intricate and diverse visual systems that maintain the distinction of being the only fully aquatic animal known to utilize the navigational strategy of path integration. They utilize idiothetic, celestial, and landmark cues to orient in the benthos. Here, we investigate the neuroanatomy of adult and developing mantis shrimp central complexes and associated neuropils to begin understanding this brain region in a sensorially and behaviorally complex crustacean.

The locus coeruleus (LC) is a small brainstem nucleus that is the primary source of noradrenaline (NA) throughout the vertebrate brain. Understanding the anatomical organization of the LC is important because NA plays a central role in regulating arousal, attention, and performance. In the mammalian brain, individual LC neurons make highly divergent axonal projections to different brain regions, which are distinguished in part by which NA receptor subtypes they express. Here we sought to determine whether similar organizational features - divergent LC projections acting regionally through different receptor subtypes - characterize cortico-basal ganglia (CBG) circuitry important to birdsong learning and performance. Single and dual retrograde tracer injections reveal that single LC-NA neurons make divergent projections to both cortical and basal ganglia components of this circuit, as well as to dopaminergic nuclei that innervate this circuit. Moreover, in situ hybridization revealed that differential expression of the 2A and 2C adrenoreceptor distinguish LC-recipient song nuclei. Therefore, LC - NA signaling in the songbird CBG circuit could employ a similar strategy as in mammals, which allows a relatively small number of LC neurons to exert widespread yet distinct effects across multiple brain regions. Key PointsO_LIThe locus coeruleus projects to most of the song system (HVC, RA, LMAN, Area X, DLM). C_LIO_LINoradrenergic receptors are regionally specialized in BG despite divergent connectivity of LC neurons. C_LIO_LINoradrenergic projections to dopaminergic nuclei could influence vocal variability and learning. C_LI

The amygdala is a complex brain structure in the vertebrate telencephalon, essential for regulating social behaviors, emotions and (social) cognition. In contrast to the vast majority of neuron types described in the many nuclei of the mammalian amygdala, little is known about the neuronal diversity in non-mammals, making reconstruction of its evolution particularly difficult. Here, we characterize glutamatergic neuron types in the amygdala of the salamander Pleurodeles waltl. Our single-cell RNA sequencing data indicate the existence of at least ten distinct types and subtypes of glutamatergic neurons in the salamander amygdala. In situ hybridization for marker genes indicates that these neuron types are located in three major subdivisions: the lateral amygdala, the medial amygdala, and a newly-defined area demarcated by high expression of the transcription factor Sim1. The gene expression profiles of these neuron types suggest similarities with specific neuron types in the sauropsid and mammalian amygdala, and in particular the evolutionary conservation of Sim1-expressing amygdalar neurons in tetrapods. Taken together, our results reveal a surprising diversity of glutamatergic neuron types in the amygdala of salamanders, despite the anatomical simplicity of their brain.

In spinal cord and medulla, ependymal cells re organized in a monolayer forming the central canal (cc). In rodents, this region, also known as a stem cell niche, was shown to contain cerebrospinal fluid-contacting neurons (CSF-cNs). These neurons are GABAergic and because of their chemo- and mechanosensory properties they would represent a novel sensory system intrinsic to the central nervous system. In primates, little is known about these neurons and more generally about the region around the cc. Here, using immunohistochemical approaches, we investigated the organization of the cc region and CSF-cN properties in Macaca mulatta Rhesus monkey. In contrast to rodent, we observe along the whole medullo-spinal axis a large zone around the cc delimited by long radial ependymal fibers that is enriched with astrocytes and microglia but largely devoid of neuronal elements except for CSF-cNs. These primate CSF-cNs share with rodent CSF-cNs similar morphological and phenotypical features with a largely immature profile. Our data suggest that they extend their axons in the longitudinal axis to form fiber bundles close to the cc and we further show that CSF-cNs receive GABAergic and serotoninergic synaptic contacts on their soma and dendrite. Taken together our results reveal in Rh. monkey a specific organization of the region around the cc potentially forming a buffer zone between CSF and parenchyma where CSF-cNs would play a crucial role in the detection of CSF signals and their transmission to the central nervous system, a role that would need to be further investigated.

The cortico-basal ganglia pathways that mediate vocal learning in zebra finches (Taeniopygia guttata) are localized in parallel circuits formed by CORE and SHELL subregions. These circuits traverse a specialized region of the basal ganglia essential for vocal learning (Area X), which includes intermixed striatal and pallidal neurons. The pallidal neurons within Area X exhibit analogs of mammalian direct and indirect pathways that may have opposing effects and thereby increase or inhibit thalamic activity respectively. Direct pallidal neurons of Area X send projections to the medial portion of the dorsolateral anterior thalamic nucleus (DLM), whereas indirect pallidal neurons form intrinsic connections onto DLM-projecting neurons. Expression of the transcription factor FoxP2 in the basal ganglia is necessary for normal vocal learning and production in both humans and songbirds. We used tract-tracing techniques to label direct pallidal Area X[->]DLM projection neurons and immunohistochemical techniques to label neurons expressing the transcription factor FoxP2 in adult and juvenile male zebra finches. Our results showed that DLM-projecting neurons did not express FoxP2 in either adults or juveniles. Measurements of nuclear sizes revealed a population of large neurons that expressed FoxP2 but were not retrogradely-labeled from DLM. A putative marker of striatal neurons (DARPP-32) did not co-localize with FoxP2 in many of these large neurons, suggesting that they form a class of indirect pallidal neurons. These findings offer FoxP2 as a possible marker for indirect pallidal neurons and support the existence of different subpopulations of neurons that correspond to direct and indirect pathways within Area X.

The intermediate and deep layers of the midbrain superior colliculus (SC) are a key locus for several critical functions, including spatial attention, multisensory integration and behavioral responses. While the SC is known to integrate input from a variety of brain regions, progress in understanding how these inputs contribute to SC-dependent functions has been hindered by the paucity of data on innervation patterns to specific types of SC neurons. Here, we use G-deleted rabies virus-mediated monosynaptic tracing to identify inputs to excitatory and inhibitory neurons of the intermediate and deep SC. We observed stronger and more numerous projections to excitatory than inhibitory SC neurons. However, a subpopulation of excitatory neurons thought to mediate behavioral output received weaker inputs, from far fewer brain regions, than the overall population of excitatory neurons. Additionally, extrinsic inputs tended to target rostral excitatory and inhibitory SC neurons more strongly than their caudal counterparts, and commissural SC neurons tended to project to similar rostrocaudal positions in the other SC. Our findings support the view that active intrinsic processes are critical to SC-dependent functions, and will enable the examination of how specific inputs contribute to these functions.

Understanding the synaptic characteristics of each cortical layer is essential for elucidating the functional architecture of each brain region. In the current study, we made a detailed quantitative comparison of the synaptic structure in the predominantly input layers of primate primary visual cortex (layer 4C) and in the predominant output layer (layer 3B) using focused ion beam scanning electron microscopy (FIB/SEM). We quantified the synaptic density in each layer, classified synaptic boutons according to their number of synapses and mitochondrial content, and quantified key morphometric parameters, including bouton volume, postsynaptic density (PSD) area and morphology, volume occupied by mitochondria, and postsynaptic targets. Our results revealed that for all the layers there is a higher proportion of single-synapse boutons without mitochondria. Multisynaptic boutons containing mitochondria (MSBm+)-- which likely correspond to TC terminals --were significantly more abundant in the thalamocortical recipient layers 4C and 4C{beta}. These MSBm+ boutons were also larger, more likely to contact dendritic spines, and contained more mitochondria than other bouton categories. In contrast, layer 3B, displayed a lower prevalence of MSBm+ boutons, these boutons were smaller than those in layer 4C and made fewer synapses. These findings highlight laminar differences in bouton architecture and support the idea that TC synapses are structurally adapted to support high synaptic efficacy. Together, our data provide a detailed quantitative framework for understanding the synaptic organization of primate V1, with implications for sensory processing and cortical circuit function.

To better understand the cortical connections and organization of visual areas in galagos, we examined the interconnections among cortical visual fields and their relationships with posterior parietal cortex (PPC) and temporal regions associated with dorsal and ventral streams of visual processing. In five galagos, two to four distinguishable tracers were injected into different visual areas, allowing direct comparison of connection patterns within the same cases. To reveal distributions of labeled neurons for each injection, labeled cells were plotted from serial brain sections cut parallel to the flattened cortical surface and summed across sections to generate surface reconstructions. Alternate sections processed for cytoarchitectonic features were used to identify cortical borders, especially those of V1 and middle temporal visual area (MT). Overall, our results support the conclusion that regions of V2 and V3 represent the contralateral visual hemifield in parallel with V1 and with each other. However, dorsal V3, representing the lower visual hemifield, includes at least one discontinuity where representations of the upper visual field extend to the dorsal border of V2. This portion of V3 appears to belong to the dorsomedial visual area (DM), which extends rostrally from V2 into PPC. The dorsal part of the DL-V4 region receives projections from other parts of dorsolateral visual area (DL), central V1, V2 and V3, inferotemporal (IT) cortex, the MT complex, and PPC regions surrounding the intraparietal sulcus (IPS). More central portions of DL-V4 receive inputs from central representations of V1, V2, and V3, as well as from PPC regions lateral to the IPS, the MT complex, and upper IT cortex. The ventral part of DL receives projections from central V2, caudal PPC adjoining DM and ventral PPC, and from IT cortex. These patterns indicate that the DL-V4 region serves as a major node linking dorsal and ventral streams and likely includes more than one functionally distinct visual area. In addition, areas MT and DM show strong reciprocal connections with PPC, while the connections of IT cortex indicate that much of this region is visual in nature having strong connections with higher order visual areas and it is composed of multiple functionally specialized visual domains. Key pointsO_LIOrganization of the visual cortex in galagos is much like that in New World and Old Word monkeys. C_LIO_LIPatterns of cortical connections of early visual areas V1 and V2 support the view that dorsal V3 has a gap in the representation of the lower visual field that is occupied by the proposed dorsomedial visual area (DM). C_LIO_LIVisual areas DM and middle temporal visual area (MT) provide the major visual inputs to posterior parietal cortex (PPC) of the dorsal stream of visual processing for actions. C_LI

Corticotropin-releasing factor (CRF) is best known for its involvement in peripheral glucocorticoid release across vertebrate species. However, CRF is also produced and released throughout various brain regions to regulate central aspects of the stress response. While these various CRF populations have been described extensively in mammals, less is known about their distributions in other amniotes, and only a handful of studies have ever examined CRF distributions in reptiles. Out study is the first to map CRF cell and fiber distributions in the brain of a lizard, the brown anole (Anolis sagrei). Our results indicate that brown anole CRF distributions are highly similar to those in snakes and turtles. However, unlike in these other reptile species, we find immunofluorescent CRF neurons in a few additional brown anole locations, most notably the supraoptic nucleus. The CRF distribution in the present study is also similar to published CRF descriptions in mammals and birds, although our findings, as well as the other published reports in reptiles, collectively suggest that reptiles possess a slightly more restricted distribution of CRF cell populations than do mammals and birds.

The mouse retina is made-up of approximately 150 types of neurons each with unique characteristics and functions in interpreting visual information. Recent efforts to categorize cell types using molecular markers, morphology, and electrophysiological response properties have provided a wealth of information and a host of tools for studying specific cell types. AAV-based approaches have several advantages over transgenic mouse lines, including ease of application to many different animal models without extensive crossing and their amenability to intersectional approaches. Here, we provide an in-depth characterization of retinal ganglion cell types labeled by two AAV vectors drawn from a recent panel of constructs with synthetic promoters. Each promoter analyzed here was derived from a gene expressed in a cell type specific manner. Using a combination of morphology, molecular markers, and electrophysiological measurement of light responses, we found that each vector labeled distinct subsets of RGCs. However, both labeled more cell types than expected from the expression pattern of the promoters endogenous gene. We then characterized the projection patterns of these RGC types to the brain, finding that each AAV type labeled distinct axonal populations. These tools provide new access to a unique subset of cells and will be instrumental to future studies analyzing their functions and connectivity.

The vomeronasal system (VNS) is critical for detecting pheromonal cues that modulate sociosexual behaviors. Despite its central role in chemical communication, our understanding of its anatomical and functional variability across mammals remains incomplete. This study provides the first detailed characterization of the VNS in the Iberian mole (Talpa occidentalis), a fossorial species endemic to the Iberian Peninsula. We performed a morphofunctional and neurochemical analysis of the vomeronasal organ (VNO) and the accessory olfactory bulb (AOB) using histology, immunohistochemistry, and lectin histochemistry. The VNO in T. occidentalis exhibited an unusual circular lumen lined by a uniform sensory epithelium, lacking the dual epithelial organization seen in most species. The vomeronasal cartilage was limited in extent and did not form the typical J-shaped structure. Importantly, no evidence of a vomeronasal pump was found, suggesting alternative mechanisms for semiochemical entry, likely facilitated by the organs anatomical position and continuous receptor distribution. Immunohistochemical analysis revealed strong expression of Gi2 and G{Upsilon}8 in sensory neurons, with weaker G0 expression, suggesting predominance of V1R-type signal transduction. The AOB, though small, exhibited clear lamination and specific marker localization (Gi2, OMP, CR, MAP2), indicating robust functional organization. Lectin binding revealed specific glycosylation patterns in the glomerular layer, with STL and LEA marking synaptic regions. These findings uncover unprecedented anatomical and molecular features in the VNS of T. occidentalis, positioning this species as a valuable model for studying vomeronasal diversity and evolution among Laurasiatherian mammals.

Accurate anatomical characterizations are necessary to investigate neural circuitry on a fine scale, but for the rodent claustrum complex (CC) this has yet to be fully accomplished. The CC is generally considered to comprise two major subdivisions, the claustrum (CL) and the dorsal endopiriform nucleus (DEn), but regional boundaries to these areas are highly debated. To address this, we conducted a multifaceted analysis of fiber- and cyto-architecture, genetic marker expression, and connectivity using mice of both sexes, to create a comprehensive guide for identifying and delineating borders to the CC. We identified four distinct subregions within the CC, subdividing both the CL and the DEn into two. Additionally, we conducted brain-wide tracing of inputs to the entire CC using a transgenic mouse line. Immunohistochemical staining against myelin basic protein (MBP), parvalbumin (PV), and calbindin (CB) revealed intricate fiber-architectural patterns enabling precise delineations of the CC and its subregions. Myelinated fibers were abundant in dorsal parts of the CL but absent in ventral parts, while parvalbumin labelled fibers occupied the entire CL. Calbindin staining revealed a central gap within the CL, which was also visible at levels anterior to the striatum. Furthermore, cells in the CL projecting to the retrosplenial-cortex were located within the myelin sparse area. By combining our own experimental data with digitally available datasets of gene expression and input connectivity, we could demonstrate that the proposed delineation scheme allows anchoring of datasets from different origins to a common reference framework. Significance statementMice are a highly tractable model for studying the claustrum complex (CC). However, without a consensus on how to delineate the CC in rodents, comparing results between studies is challenging. It is therefore important to expand our anatomical knowledge of the CC, to match the level of detail needed to study its functional properties. Using multiple strategies for identifying claustral borders, we created a comprehensive guide to delineate the CC and its subregions. This anatomical framework will allow researchers to anchor future experimental data into a common reference space. We demonstrated the power of this new structural framework by combining our own experimental data with digitally available data on gene expression and input connectivity of the CC.

Over the last two decades, beginning with the Avian Brain Nomenclature Forum in 2000, major revisions have been made to our understanding of the organization and nomenclature of the avian brain. However, there are still unresolved questions on avian pallial organization, particularly whether the cells above the ventricle represent different populations to those below it. Concerns included limited number of genes profiled, biased selection of genes, and potential independent origins of cell types in different parts of the brain. Here we test two competing hypotheses, using RNA sequencing to profile the transcriptomes of the major avian pallial subdivisions dorsal and ventral to the ventricle boundary, and a new zebra finch genome assembly containing about 22,000 annotated, complete genes. We found that the transcriptomes of neural populations below and above the ventricle were remarkably similar. What had been previously named hyperpallium densocellulare above the ventricle had nearly the same molecular profile as the mesopallium below it; the hyperpallium apicale above was highly similar to the nidopallium below; the primary sensory intercalated hyperpallium apicale above was most similar to the sensory population below, although more divergent than the other populations were to each other. These shared population expression profiles define unique functional specializations in anatomical structure development, synaptic transmission, signaling, and neurogenesis. These findings support the continuum hypothesis of avian brain subdivisions above and below the ventricle space, with the pallium as a whole consisting of four major cell populations instead of seven and has some profound implications for our understanding of vertebrate brain evolution.

Animals produce different vocalization types, which differ in their acoustic features and are produced in different behavioral contexts. How vocalization-related brain circuits are organized to enable the production of different vocalization types remains poorly understood. The nucleus retroambiguus is a hindbrain premotor region that regulates the production of both ultrasonic vocalizations (USVs) and distress calls (squeaks) in adult mice, but whether distinct or overlapping populations of RAm neurons are recruited during the production of these two vocalization types is unknown. In the current study, we used Fos immunohistochemistry to compare the counts and spatial distributions of Fos-positive RAm neurons in males and females that produced USVs and females that produced courtship squeaks. We also combined in vivo activity-dependent (TRAP2) labeling with Fos immunohistochemistry to directly compare Fos expression associated with the production of USVs and courtship squeaks in the same females. Our findings suggest that RAm contains three vocalization-related populations of neurons: squeak-related neurons, USV-related neurons, and shared neurons that are recruited during both vocalization types. These findings refine current models of the premotor control of vocalization and set the stage for future work to explore anatomical and functional heterogeneity within RAm.

Insect brains are formed by conserved sets of neural lineages whose fibres form cohesive bundles with characteristic projection patterns. Within the brain neuropil these bundles establish a system of fascicles constituting the macrocircuitry of the brain. The overall architecture of the neuropils and the macrocircuitry appear to be conserved. However, variation is observed e.g., in size and shape and timing of development. Unfortunately, the developmental and genetic basis of this variation is poorly understood although the rise of new genetically tractable model organisms such as the red flour beetle Tribolium castaneum allows the possibility to gain mechanistic insights. To facilitate such work, we present an atlas of the developing brain of T. castaneum, covering the first larval instar, the prepupal stage and the adult, by combining wholemount immunohistochemical labelling of fibre bundles (acetylated tubulin) and neuropils (synapsin) with digital 3D reconstruction using the TrakEM2 software package. Upon comparing this anatomical dataset with the published work in D. melanogaster, we confirm an overall high degree of conservation. Fibre tracts and neuropil fascicles, which can be visualized by global neuronal antibodies like anti-acetylated tubulin in all invertebrate brains, create a rich anatomical framework to which individual neurons or other regions of interest can be referred to. The framework of a largely conserved pattern allowed us to describe differences between the two species with respect to parameters such as timing of neuron proliferation and maturation. These features likely reflect adaptive changes in developmental timing that govern the change from larval to adult brain.

The functional organization of the teleost telencephalic pallium remains poorly understood, particularly regarding the presence of modality-specific sensory domains and their topographic arrangement. Here, we used in vivo wide-field voltage-sensitive dye imaging to map sensory-evoked neural activity across the dorsal surface of the telencephalic pallium of adult goldfish. Somatosensory, auditory, gustatory, and visual stimulation revealed distinct, modality-specific domains primarily located within the dorsomedial (Dm) and dorsolateral (Dl) pallium. Within Dm, somatosensory and auditory stimuli activated partially overlapping territories in the caudal subregion (Dm4), exhibiting clear somatotopic and tonotopic organization along the mediolateral axis. Gustatory stimulation selectively engaged Dm3, where different tastants activated spatially distinct but partially overlapping domains. A more rostral subregion (Dm2) responded only to high-intensity somatosensory stimulation, suggesting involvement in processing negatively valenced inputs. Visual stimulation activated a circumscribed area within the dorsolateral pallium (Dld2),that closely matched cytoarchitectural boundaries. Pharmacological blockade of ionotropic glutamate receptors markedly reduced sensory-evoked responses, indicating that these maps depend on glutamatergic synaptic transmission. Together, these findings show that the goldfish pallium contains distinct, spatially organized sensory representations and a refined internal functional architecture. This organization suggests that pallial topographic sensory maps may not be exclusive to mammals and birds. Based on these results, we propose that dorsomedial and dorsolateral pallial regions may be functionally comparable to components of the mammalian mesocortical network, more than to the pallial amygdala or the neocortex. This framework provides a new perspective on pallial organization in teleosts and contributes to understanding the evolutionary origins of the vertebrate pallium. HIGHLIGHTSO_LIVoltage-sensitive dye imaging was used to map sensory responses in the goldfish pallium. C_LIO_LIDistinct sensory areas for somatosensory, auditory, gustatory, and visual modalities were identified. C_LIO_LISome sensory regions in Dm show topographically organized maps. C_LIO_LIFunctional segregation suggests a complex, non-diffuse pallial organization. C_LIO_LIFindings support a novel hypothesis linking Dm and Dld to mammalian mesocortical regions. C_LI

We recently investigated whether activity in primary visual cortex of a primate (Callithrix jacchus) is modulated during running, and found that the effects were small (and suppressive), a notable difference from the large and positive modulations observed in mice. In that first report, we noted that the majority of our data were collected from the retinotopic representation of the fovea, and surmised that running modulations might be different in the peripheral representation. Here, we report that running-correlated modulations of the peripheral representation in marmoset V1 are positive and substantial-- on order of 30%. In light of both the small and negative modulations observed in foveal V1, and the large and positive modulations seen in mouse V1, these results suggest that the foveal representation in primates may be unique. In this domain, non-foveal V1 in primates appears more similar to that of rodents.

The mammalian olfactory systems can be divided into several subsystems based on the anatomical location of their neuroreceptor cells and the family of receptors they express. The more in depth studied systems are the main olfactory system and the vomeronasal system, whose first integrative enters are the main and the accessory olfactory bulb, respectively. In addition, there is a range of olfactory subsystems which converge to the transition zone located between the main olfactory bulb and the accessory olfactory bulb., which has been termed as olfactory limbus (OL) and includes specialized glomeruli which receive uncanonical sensory afferences and interact with the MOB and AOB. Beyond the laboratory rodents, there is a lack of information regarding the olfactory subsystems of carnivores. We have focused on the specific study of the olfactory limbus of the fox, performing serial histological sections, general and specific histological stainings, including both double and simple immunohistochemical and lectin-histochemical labeling techniques. As a result, we have been able to determine that the OL of the fox shows an uncommon development with a high degree of development and complexity. This makes this species a novel mammalian model that could provide a wider understanding of non-canonical pathways involved in the processing of chemosensory cues.

Invertebrate chordates, such as the tunicate Ciona, can offer insight into the evolution of the chordate phylum. Anatomical features that are shared between invertebrate chordates and vertebrates may be taken as evidence of their presence in a common chordate ancestor. The central nervous systems of Ciona larvae and vertebrates share a similar anatomy despite the Ciona CNS having [~]180 neurons. However, the depth of conservation between the Ciona CNS and those in vertebrates is not resolved. The Ciona caudal CNS, while appearing spinal cord-like, has hitherto been thought to lack motor neurons, bringing into question its homology with the vertebrate spinal cord. We show here that the Ciona larval caudal CNS does, in fact, have functional motor neurons along its length, pointing to the presence of a spinal cord-like structure at the base of the chordates. We extend our analysis of shared CNS anatomy further to explore the Ciona "motor ganglion", which has been proposed to be a homolog of the vertebrate hindbrain, spinal cord, or both. We find that a cluster of neurons in the dorsal motor ganglion shares anatomical location, developmental pathway, neural circuit architecture, and gene expression with the vertebrate cerebellum. However, functionally, the Ciona cluster appears to have more in common with vertebrate cerebellum-like structures, insofar as it receives and processes direct sensory input. These findings are consistent with earlier speculation that the cerebellum evolved from a cerebellum-like structure, and suggest that the latter structure was present in the dorsal hindbrain of a common chordate ancestor.

The mammalian visual system consists of two distinct pathways: rod- and cone-driven vision. The rod pathway is responsible for dim light vision whereas the cone pathway mediates daylight vision and color perception. The distinct processing of visual information begins at the first synapse of rod and cone photoreceptors. The unique composition and organization of the rod and cone synapse is what allows information to be parsed into the different visual pathways. Although this is a critical process for vision, little is known about the key molecules responsible for establishing and maintaining the distinct synaptic architecture of the rod and cone synapse. In the present study, we uncovered a new role for Ankyrins in maintaining the synaptic integrity of the rod and cone synapse. Loss of Ankyrin-B and Ankyrin-G results in connectivity defects between photoreceptors and their synaptic partners. Ultrastructure analysis of the rod and cone synapse revealed impaired synaptic innervation, abnormal terminal morphology, and disruption of synaptic connections. Consistent with these findings, functional studies revealed impaired in vivo retinal responses in animals with loss of Ankyrin-B and Ankyrin-G. Taken together, our data supports a new role for Ankyrins in maintaining synaptic integrity and organization of photoreceptor synapses in the mouse outer retina. SIGNFICANCE STATEMENTThe first synapse in the outer retina begins to process visual information into two distinct pathways. This is largely attributed to the different composition and organization of the rod and cone synapse. Although the structural integrity of the rod and cone synapse is critical for normal vision, little is known about the key molecules responsible for maintaining the unique structure of the different photoreceptor synapses. In this study, we demonstrate a new function for the cytoskeletal scaffolding proteins, Ankryin-B and Ankyrin-G in the mouse outer retina. We found Ankyrin-B and Ankyrin-G are both required for proper retinal connectivity, where loss of these molecules leads to synaptic defects and impaired retinal responses.