Introduction: Accumulation of β2-microglobulin (B2M) in dialysis patients contributes to several comorbidities of end stage kidney disease (ESKD). The LIXELLE® device adsorbs B2M from blood using sorbent bead technology. Studies in Japan showed LIXELLE treatment during hemodialysis (HD) at blood flow rates up to 250 mL/min removes B2M above HD alone and is well-tolerated. We investigated tolerance for LIXELLE treatment during HD at higher HD blood flow rates standard in the United States (US). Methods: A prospective, open-label, non-randomized, single arm, early feasibility study (EFS) assessed tolerance and safety of LIXELLE treatment during HD at blood flow rates up to 450 mL/min. ESKD patients (40-75 years old) on thrice weekly outpatient HD were eligible. After a one-week HD run-in, patients received LIXELLE plus HD at a blood flow rate of 250 mL/min (one week), followed by LIXELLE plus HD at a blood flow rate up to 450 mL/min (one week). These blood flow rates were tested with three LIXELLE column sizes in sequence (treatment = six weeks). B2M removal was assessed for each combination. Results: Ten patients with a historic intradialytic hypotension (IDH) rate of 0.42 events/HD session/patient were enrolled. Nine patients completed all combinations without IDH events (treatment IDH rate 0.56 events/HD session/patient). No treatment-emergent serious adverse events or significant changes in red blood cell, platelet, or complement indices except haptoglobin were reported. B2M reduction ratios and removal of select proteins (<40 kDa) increased with escalating column size and blood flow rate. Conclusion: LIXELLE plus HD across all column sizes was safe and well-tolerated at blood flow rates up to 450 mL/min. Extent of B2M removal corresponded to column size-blood flow rate combinations. This EFS provides a risk profile to guide further studies of LIXELLE in ESKD patients at US-standard blood flow rates.

C-C chemokine receptor type 3 (CCR3) has been linked with age-related macular degeneration (AMD) pathologies. Specifically, its function as an immune modulator in AMD remains unclear. To address this question, we investigated the impact of CCR3 inhibition on inflammation, a key driver of AMD pathologies, by assessing inflammatory cytokines and infiltrating immune cells in two models of ocular inflammation. Mice were orally dosed twice a day with AKST4290, a CCR3 small molecular inhibitor, in the sodium iodate (NaIO3) and myelin oligodendrocyte glycoprotein (MOG) models. A combination of autoradiography and analytical chemistry techniques were used to assess drug concentration and distribution. Bead-based multiplexing technology was used to determine cytokine concentrations, and flow cytometry and immunohistochemistry was used to ascertain ocular immune-cell composition. CCR3 expression was detected in the retinal pigment epithelium (RPE)/choroid complex where AKST4290 was found to preferentially accumulate at sustained levels. In the NaIO 3 model, inhibition of CCR3 with AKST4290 significantly decreased both the concentration of specific chemokines and the number of multiple populations of infiltrating peripheral immune cell. Furthermore, effects of CCR3 inhibition on immune cell infiltration were confirmed in the MOG model. These data demonstrate that CCR3 inhibition strongly modulates local inflammation by impacting both cytokine concentrations and immune cell composition in ocular diseases. Moreover, these findings together with the known role of CCR3 in promoting pathologic angiogenesis implicate a pleiotropic role for CCR3 in AMD.

The wiring of visual circuits requires that retinal neurons functionally connect to specific brain targets, a process that involves activity-dependent signaling between retinal axons and their postsynaptic targets. Vision loss in various ophthalmological and neurological diseases is caused by damage to the connections from the eye to the brain. How postsynaptic brain targets influence retinal ganglion cell (RGC) axon regeneration and functional reconnection with the brain targets remains poorly understood. Here, we established a paradigm in which the enhancement of neural activity in the distal optic pathway, where the postsynaptic visual target neurons reside, promotes RGC axon regeneration and target reinnervation and leads to the rescue of optomotor function. Furthermore, selective activation of retinorecipient neuron subsets is sufficient to promote RGC axon regeneration. Our findings reveal a key role for postsynaptic neuronal activity in the repair of neural circuits and highlight the potential to restore damaged sensory inputs via proper brain stimulation.

Targeting immune-mediated, age-related, biology has the potential to be a transformative therapeutic strategy. However, the redundant nature of the multiple cytokines that change with aging requires identification of a master downstream regulator to successfully exert therapeutic efficacy. Here, we discovered CCR3 as a prime candidate, and inhibition of CCR3 has pro-cognitive benefits in mice, but these benefits are not driven by an obvious direct action on central nervous system (CNS)-resident cells. Instead, CCR3-expressing T cells in the periphery that are modulated in aging inhibit infiltration of these T cells across the blood-brain barrier and reduce neuroinflammation. The axis of CCR3-expressing T cells influencing crosstalk from periphery to brain provides a therapeutically tractable link. These findings indicate the broad therapeutic potential of CCR3 inhibition in a spectrum of neuroinflammatory diseases of aging.

Loss of retinal ganglion cells (RGCs) underlies several forms of retinal disease including glaucomatous optic neuropathy, a leading cause of irreversible blindness. Several rare genetic disorders associated with cilia dysfunction have retinal degeneration as a clinical hallmark. Much of the focus of ciliopathy associated blindness is on the connecting cilium of photoreceptors; however, RGCs also possess primary cilia. It is unclear what roles RGC cilia play, what proteins and signaling machinery localize to RGC cilia, or how RGC cilia are differentiated across the subtypes of RGCs. To better understand these questions, we assessed the presence or absence of a prototypical cilia marker Arl13b and a widely distributed neuronal cilia marker AC3 in different subtypes of mouse RGCs. Interestingly, not all RGC subtype cilia are the same and there are significant differences even among these standard cilia markers. Alpha-RGCs positive for osteopontin, calretinin, and SMI32 primarily possess AC3-positive cilia. Directionally selective RGCs that are CART positive or Trhr positive localize either Arl13b or AC3, respectively, in cilia. Intrinsically photosensitive RGCs differentially localize Arl13b and AC3 based on melanopsin expression. Taken together, we characterized the localization of gold standard cilia markers in different subtypes of RGCs and conclude that cilia within RGC subtypes may be differentially organized. Future studies aimed at understanding RGC cilia function will require a fundamental ability to observe the cilia across subtypes as their signaling protein composition is elucidated. A comprehensive understanding of RGC cilia may reveal opportunities to understanding how their dysfunction leads to retinal degeneration.

The authors have requested that this preprint be removed from Research Square.

The mammalian visual system is composed of circuitry connecting sensory input from the retina to the processing core of the visual cortex. The two main retinorecipient brain targets, the superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN), bridge retinal input and visual output. The primary cilium is a conserved organelle increasingly viewed as a critical sensor for the regulation of developmental and homeostatic pathways in most mammalian cell types. Moreover, cilia have been described as crucial for neurogenesis, neuronal maturation, and survival in the cortex and retina. However, cilia in the visual relay center remain to be fully described. In this study, we characterized the ciliation profile of the SC and dLGN and found that the overall number of ciliated cells declined during development. Interestingly, shorter ciliated cells in both regions were identified as neurons, whose numbers remained stable over time, suggesting that cilia retention is a critical feature for optimal neuronal function in SC and dLGN. Our study suggests that primary cilia are important for neuronal maturation and function in cells of the SC and dLGN.

The functional attributes of parallel sensory circuits are determined by the properties of the individual cell types that comprise them, and their connectivity. The mechanisms controlling the specification and establishment of parallel sensory pathways remain unclear. Here we report the non-uniform distribution of retinal ganglion cells (RGCs) expressing the transcription factor Tbx20 in mice. Owing to their layout within the retina, Tbx20-RGCs are positioned to preferentially encode light information from dorsal visual space. We discovered that Tbx20 expression is mostly restricted to three RGC-types: M1 intrinsically photosensitive RGCs (ipRGCs), M2 ipRGCs and 'diving' RGCs. The axonal projections of Tbx20-RGCs innervate non-image-forming brain centers including the suprachiasmatic nucleus, the medial division of the posterior pretectal nucleus, and the olivary pretectal nucleus (OPN), a principal station in the neural pathway for generating the pupillary light reflex (PLR). Conditional deletion of Tbx20 resulted in reduced Tbx20-RGC axonal innervation of these targets and revealed a key role of these neurons in driving specific phases of the PLR. Furthermore, chemogenetic activation uncovered a novel role for a specific subset of Tbx20-RGCs in controlling pupil dilation. These data offer a new understanding of the genetic and molecular mechanisms that establish specific, behaviorally-relevant visual circuits.

The ability to detect moving objects is an ethologically salient function. Direction-selective neurons have been identified in the retina, thalamus, and cortex of many species, but their homology has remained unclear. For instance, it is unknown whether direction-selective retinal ganglion cells (DSGCs) exist in primates and, if so, whether they are the equivalent to mouse and rabbit DSGCs. Here, we used a molecular/circuit approach in both sexes to address these issues. In mice, we identify the transcription factor Satb2 (special AT-rich sequence-binding protein 2) as a selective marker for three RGC types: On–Off DSGCs encoding motion in either the anterior or posterior direction, a newly identified type of Off-DSGC, and an Off-sustained RGC type. In rabbits, we find that expression of Satb2 is conserved in On–Off DSGCs; however, it has evolved to include On–Off DSGCs encoding upward and downward motion in addition to anterior and posterior motion. Next, we show that macaque RGCs express Satb2 most likely in a single type. We used rabies virus-based circuit-mapping tools to reveal the identity of macaque Satb2-RGCs and discovered that their dendritic arbors are relatively large and monostratified. Together, these data indicate Satb2-expressing On–Off DSGCs are likely not present in the primate retina. Moreover, if DSGCs are present in the primate retina, it is unlikely that they express Satb2. SIGNIFICANCE STATEMENT The ability to detect object motion is a fundamental feature of almost all visual systems. Here, we identify a novel marker for retinal ganglion cells encoding directional motion that is evolutionarily conserved in mice and rabbits, but not in primates. We show in macaque monkeys that retinal ganglion cells (RGCs) that express this marker comprise a single type and are morphologically distinct from mouse and rabbit direction-selective RGCs. Our findings indicate that On–Off direction-selective retinal neurons may have evolutionarily diverged in primates and more generally provide novel insight into the identity and organization of primate parallel visual pathways.

The use of sensory information to drive specific behaviors relies on circuits spanning long distances that wire up through a range of axon-target recognition events. Mechanisms assembling poly-synaptic circuits and the extent to which parallel pathways can "cross-wire" to compensate for loss of one another remain unclear and are crucial to our understanding of brain development and models of regeneration. In the visual system, specific retinal ganglion cells (RGCs) project to designated midbrain targets connected to downstream circuits driving visuomotor reflexes. Here, we deleted RGCs connecting to pupillary light reflex (PLR) midbrain targets and discovered that axon-target matching is tightly regulated. RGC axons of the eye-reflex pathway avoided vacated PLR targets. Moreover, downstream PLR circuitry is maintained; hindbrain and peripheral components retained their proper connectivity and function. These findings point to a model in which poly-synaptic circuit development reflects independent, highly stringent wiring of each parallel pathway and downstream station.

Accurate motion detection requires neural circuitry that compensates for global visual field motion. Select subtypes of retinal ganglion cells perceive image motion and connect to the accessory optic system (AOS) in the brain, which generates compensatory eye movements that stabilize images during slow visual field motion. Here, we show that the murine transmembrane semaphorin 6A (Sema6A) is expressed in a subset of On direction-selective ganglion cells (On DSGCs) and is required for retino-recipient axonal targeting to the medial terminal nucleus (MTN) of the AOS. Plexin A2 and A4, two Sema6A binding partners, are expressed in MTN cells, attract Sema6A(+) On DSGC axons, and mediate MTN targeting of Sema6A(+) RGC projections. Furthermore, Sema6A/Plexin-A2/A4 signaling is required for the functional output of the AOS. These data reveal molecular mechanisms underlying the assembly of AOS circuits critical for moving image perception.

There are many transgenic GFP reporter lines that allow the visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. We developed a method that exploits GFP for gene manipulation, Cre recombinase dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, which led to effective recombination selectively in GFP-labeled cells. Furthermore, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+cells, we found that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.

Every aspect of visual perception and behavior is built from the neural activity of retinal ganglion cells (RGCs), the output neurons of the eye. Here, we review progress toward understanding the many types of RGCs that communicate visual signals to the brain, along with the subcortical brain regions that use those signals to build and respond to representations of the outside world. We emphasize recent progress in the use of mouse genetics, viral circuit tracing, and behavioral psychophysics to define and map the various RGCs and their associated networks. We also address questions about the homology of RGC types in mice and other species including nonhuman primates and humans. Finally, we propose a framework for understanding RGC typology and for highlighting the relationship between RGC type-specific circuitry and the processing stations in the brain that support and give rise to the perception of sight.

How specific features in the environment are represented within the brain is an important unanswered question in neuroscience. A subset of retinal neurons, called direction-selective ganglion cells (DSGCs), are specialized for detecting motion along specific axes of the visual field. Despite extensive study of the retinal circuitry that endows DSGCs with their unique tuning properties, their downstream circuitry in the brain and thus their contribution to visual processing has remained unclear. In mice, several different types of DSGCs connect to the dorsal lateral geniculate nucleus (dLGN), the visual thalamic structure that harbours cortical relay neurons. Whether direction-selective information computed at the level of the retina is routed to cortical circuits and integrated with other visual channels, however, is unknown. Here we show that there is a di-synaptic circuit linking DSGCs with the superficial layers of the primary visual cortex (V1) by using viral trans-synaptic circuit mapping and functional imaging of visually driven calcium signals in thalamocortical axons. This circuit pools information from several types of DSGCs, converges in a specialized subdivision of the dLGN, and delivers direction-tuned and orientation-tuned signals to superficial V1. Notably, this circuit is anatomically segregated from the retino-geniculo-cortical pathway carrying non-direction-tuned visual information to deeper layers of V1, such as layer 4. Thus, the mouse harbours several functionally specialized, parallel retino-geniculo-cortical pathways, one of which originates with retinal DSGCs and delivers direction- and orientation-tuned information specifically to the superficial layers of the primary visual cortex. These data provide evidence that direction and orientation selectivity of some V1 neurons may be influenced by the activation of DSGCs.

Everything the brain knows about the content of the visual world is built from the spiking activity of retinal ganglion cells (RGCs). As the output neurons of the eye, RGCs include ∼20 different subtypes, each responding best to a specific feature in the visual scene. Here we discuss recent advances in identifying where different RGC subtypes route visual information in the brain, including which targets they connect to and how their organization within those targets influences visual processing. We also highlight examples where causal links have been established between specific RGC subtypes, their maps of central connections and defined aspects of light-mediated behavior and we suggest the use of techniques that stand to extend these sorts of analyses to circuits underlying visual perception.