Molecular Fingerprinting of On-Off Direction-Selective Retinal Ganglion Cells Across Species and Relevance to Primate 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.

Rapid Detection and Monitoring of Flavobacterium psychrophilum in Water by Using a Handheld, Field-Portable Quantitative PCR System.

Advances in technology are making it easier for rapid field detection of microbes in aquaculture. Specifically, real-time quantitative PCR (qPCR) analysis, which has traditionally been confined to laboratory-based protocols, is now available in a handheld, field-portable system. The feasibility of using the Biomeme handheld qPCR system for rapid (<50 min) on-site detection and monitoring of Flavobacterium psychrophilum from filtered water samples was evaluated. Paired water samples were collected over a 23-d period from microcosm tanks that housed fish injected with known levels of F. psychrophilum. Water samples were filtered through 0.45-µm nitrocellulose filters and were analyzed with both the Biomeme qPCR platform and a traditional bench qPCR protocol. The two methods identified similar fluctuations in F. psychrophilum DNA throughout the study. Standard curves relating quantification cycles to the number of F. psychrophilum colony-forming units (CFU) were constructed and analyzed; results indicated that CFU increased rapidly between days 6 and 8 of the trial and then progressively decreased during the remaining 15 d. Average calculated log10 (CFU/mL) values were significantly correlated between the two platforms. Rapid, field-based qPCR can be incorporated into daily water quality monitoring protocols to help detect and monitor microbes in aquaculture systems.

Strict Independence of Parallel and Poly-synaptic Axon-Target Matching during Visual Reflex Circuit Assembly.

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.

Neural activity promotes long-distance, target-specific regeneration of adult retinal axons.

Axons in the mammalian CNS fail to regenerate after injury. Here we show that if the activity of mouse retinal ganglion cells (RGCs) is increased by visual stimulation or using chemogenetics, their axons regenerate. We also show that if enhancement of neural activity is combined with elevation of the cell-growth-promoting pathway involving mammalian target of rapamycin (mTOR), RGC axons regenerate long distances and re-innervate the brain. Analysis of genetically labeled RGCs revealed that this regrowth can be target specific: RGC axons navigated back to their correct visual targets and avoided targets incorrect for their function. Moreover, these regenerated connections were successful in partially rescuing a subset of visual behaviors. Our findings indicate that combining neural activity with activation of mTOR can serve as powerful tool for enhancing axon regeneration, and they highlight the remarkable capacity of CNS neurons to re-establish accurate circuit connections in adulthood.

Contactin-4 mediates axon-target specificity and functional development of the accessory optic system.

The mammalian eye-to-brain pathway includes more than 20 parallel circuits, each consisting of precise long-range connections between specific sets of retinal ganglion cells (RGCs) and target structures in the brain. The mechanisms that drive assembly of these parallel connections and the functional implications of their specificity remain unresolved. Here we show that in the absence of contactin 4 (CNTN4) or one of its binding partners, amyloid precursor protein (APP), a subset of direction-selective RGCs fail to target the nucleus of the optic tract (NOT)--the accessory optic system (AOS) target controlling horizontal image stabilization. Conversely, ectopic expression of CNTN4 biases RGCs to arborize in the NOT, and that process also requires APP. Our data reveal critical and novel roles for CNTN4/APP in promoting target-specific axon arborization, and they highlight the importance of this process for functional development of a behaviorally relevant parallel visual pathway.

Birthdate and outgrowth timing predict cellular mechanisms of axon target matching in the developing visual pathway.

How axons select their appropriate targets in the brain remains poorly understood. Here, we explore the cellular mechanisms of axon target matching in the developing visual system by comparing four transgenic mouse lines, each with a different population of genetically labeled retinal ganglion cells (RGCs) that connect to unique combinations of brain targets. We find that the time when an RGC axon arrives in the brain is correlated with its target selection strategy. Early-born, early-arriving RGC axons initially innervate multiple targets. Subsequently, most of those connections are removed. By contrast, later-born, later-arriving RGC axons are highly accurate in their initial target choices. These data reveal the diversity of cellular mechanisms that mammalian CNS axons use to pick their targets and highlight the key role of birthdate and outgrowth timing in influencing this precision. Timing-based mechanisms may underlie the assembly of the other sensory pathways and complex neural circuitry in the brain.

A dedicated circuit links direction-selective retinal ganglion cells to the primary visual cortex.

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.

Genetic dissection of retinal inputs to brainstem nuclei controlling image stabilization.

When the head rotates, the image of the visual world slips across the retina. A dedicated set of retinal ganglion cells (RGCs) and brainstem visual nuclei termed the "accessory optic system" (AOS) generate slip-compensating eye movements that stabilize visual images on the retina and improve visual performance. Which types of RGCs project to each of the various AOS nuclei remain unresolved. Here we report a new transgenic mouse line, Hoxd10-GFP, in which the RGCs projecting to all the AOS nuclei are fluorescently labeled. Electrophysiological recordings of Hoxd10-GFP RGCs revealed that they include all three subtypes of On direction-selective RGCs (On-DSGCs), responding to upward, downward, or forward motion. Hoxd10-GFP RGCs also include one subtype of On-Off DSGCs tuned for forward motion. Retrograde circuit mapping with modified rabies viruses revealed that the On-DSGCs project to the brainstem centers involved in both horizontal and vertical retinal slip compensation. In contrast, the On-Off DSGCs labeled in Hoxd10-GFP mice projected to AOS nuclei controlling horizontal but not vertical image stabilization. Moreover, the forward tuned On-Off DSGCs appear physiologically and molecularly distinct from all previously genetically identified On-Off DSGCs. These data begin to clarify the cell types and circuits underlying image stabilization during self-motion, and they support an unexpected diversity of DSGC subtypes.

Cadherin-6 mediates axon-target matching in a non-image-forming visual circuit.

Neural circuits consist of highly precise connections among specific types of neurons that serve a common functional goal. How neurons distinguish among different synaptic targets to form functionally precise circuits remains largely unknown. Here, we show that during development, the adhesion molecule cadherin-6 (Cdh6) is expressed by a subset of retinal ganglion cells (RGCs) and also by their targets in the brain. All of the Cdh6-expressing retinorecipient nuclei mediate non-image-forming visual functions. A screen of mice expressing GFP in specific subsets of RGCs revealed that Cdh3-RGCs which also express Cdh6 selectively innervate Cdh6-expressing retinorecipient targets. Moreover, in Cdh6-deficient mice, the axons of Cdh3-RGCs fail to properly innervate their targets and instead project to other visual nuclei. These findings provide functional evidence that classical cadherins promote mammalian CNS circuit development by ensuring that axons of specific cell types connect to their appropriate synaptic targets.

Transgenic mice reveal unexpected diversity of on-off direction-selective retinal ganglion cell subtypes and brain structures involved in motion processing.

On-Off direction-selective retinal ganglion cells (DSGCs) encode the axis of visual motion. They respond strongly to an object moving in a preferred direction and weakly to an object moving in the opposite, "null," direction. Historically, On-Off DSGCs were classified into four subtypes according to their directional preference (anterior, posterior, superior, or inferior). Here, we compare two genetically identified populations of On-Off DSGCs: dopamine receptor 4 (DRD4)-DSGCs and thyrotropin-releasing hormone receptor (TRHR)-DSGCs. We find that although both populations are tuned for posterior motion, they can be distinguished by a variety of physiological and anatomical criteria. First, the directional tuning of TRHR-DSGCs is broader than that of DRD4-DSGCs. Second, whereas both populations project similarly to the dorsal lateral geniculate nucleus, they project differently to the ventral lateral geniculate nucleus and the superior colliculus. Moreover, TRHR-DSGCs, but not DRD4-DSGCs, also project to the zona incerta, a thalamic area not previously known to receive direction-tuned visual information. Our findings reveal unexpected diversity among mouse On-Off DSGC subtypes that uniquely process and convey image motion to the brain.