The retina's neurovascular unit: Müller glial sheaths and neuronal contacts.

The neurovascular unit (NVU), comprising vascular, glial and neural elements, supports the energetic demands of neural computation, but this aspect of the retina's trilaminar vessel network is poorly understood. Only the innermost vessel layer - the superficial vascular plexus (SVP) - is ensheathed by astrocytes, like brain capillaries, whereas glial ensheathment in other layers derives from radial Müller glia. Using serial electron microscopy reconstructions from mouse and primate retina, we find that Müller processes cover capillaries in a tessellating pattern, mirroring the tiled astrocytic endfeet wrapping brain capillaries. However, gaps in the Müller sheath, found mainly in the intermediate vascular plexus (IVP), permit different neuron types to contact pericytes and the endothelial cells directly. Pericyte somata are a favored target, often at spine-like structures with a reduced or absent vascular basement lamina. Focal application of adenosine triphosphate (ATP) to the vitreal surface evoked Ca2+ signals in Müller sheaths in all three vascular layers. Pharmacological experiments confirmed that Müller sheaths express purinergic receptors that, when activated, trigger intracellular Ca2+ signals that are amplified by IP3-controlled intracellular Ca2+ stores. When rod photoreceptors die in a mouse model of retinitis pigmentosa (rd10), Müller sheaths dissociate from the deep vascular plexus (DVP) but are largely unchanged within the IVP or SVP. Thus, Müller glia interact with retinal vessels in a laminar, compartmentalized manner: glial sheathes are virtually complete in the SVP but fenestrated in the IVP, permitting direct neural-to-vascular contacts. In the DVP, the glial sheath is only modestly fenestrated and is vulnerable to photoreceptor degeneration.

A circuit suppressing retinal drive to the optokinetic system during fast image motion.

Optokinetic nystagmus (OKN) assists stabilization of the retinal image during head rotation. OKN is driven by ON direction selective retinal ganglion cells (ON DSGCs), which encode both the direction and speed of global retinal slip. The synaptic circuits responsible for the direction selectivity of ON DSGCs are well understood, but those sculpting their slow-speed preference remain enigmatic. Here, we probe this mechanism in mouse retina through patch clamp recordings, functional imaging, genetic manipulation, and electron microscopic reconstructions. We confirm earlier evidence that feedforward glycinergic inhibition is the main suppressor of ON DSGC responses to fast motion, and reveal the source for this inhibition-the VGluT3 amacrine cell, a dual neurotransmitter, excitatory/inhibitory interneuron. Together, our results identify a role for VGluT3 cells in limiting the speed range of OKN. More broadly, they suggest VGluT3 cells shape the response of many retinal cell types to fast motion, suppressing it in some while enhancing it in others.

Distinctive synaptic structural motifs link excitatory retinal interneurons to diverse postsynaptic partner types.

Neurons make converging and diverging synaptic connections with distinct partner types. Whether synapses involving separate partners demonstrate similar or distinct structural motifs is not yet well understood. We thus used serial electron microscopy in mouse retina to map output synapses of cone bipolar cells (CBCs) and compare their structural arrangements across bipolar types and postsynaptic partners. Three presynaptic configurations emerge-single-ribbon, ribbonless, and multiribbon synapses. Each CBC type exploits these arrangements in a unique combination, a feature also found among rabbit ON CBCs. Though most synapses are dyads, monads and triads are also seen. Altogether, mouse CBCs exhibit at least six motifs, and each CBC type uses these in a stereotypic pattern. Moreover, synapses between CBCs and particular partner types appear biased toward certain motifs. Our observations reveal synaptic strategies that diversify the output within and across CBC types, potentially shaping the distinct functions of retinal microcircuits.

Luxotonic signals in human prefrontal cortex as a possible substrate for effects of light on mood and cognition.

Studies with experimental animals have revealed a mood-regulating neural pathway linking intrinsically photosensitive retinal ganglion cells (ipRGCs) and the prefrontal cortex (PFC), involved in the pathophysiology of mood disorders. Since humans also have light-intensity-encoding ipRGCs, we asked whether a similar pathway exists in humans. Here, functional MRI was used to identify PFC regions and other areas exhibiting light-intensity-dependent signals. We report 26 human brain regions having activation that either monotonically decreases or monotonically increases with light intensity. Luxotonic-related activation occurred across the cerebral cortex, in diverse subcortical structures, and in the cerebellum, encompassing regions with functions related to visual image formation, motor control, cognition, and emotion. Light suppressed PFC activation, which monotonically decreased with increasing light intensity. The sustained time course of light-evoked PFC responses and their susceptibility to prior light exposure resembled those of ipRGCs. These findings offer a functional link between light exposure and PFC-mediated cognitive and affective phenomena.

Spatially displaced excitation contributes to the encoding of interrupted motion by a retinal direction-selective circuit.

Spatially distributed excitation and inhibition collectively shape a visual neuron's receptive field (RF) properties. In the direction-selective circuit of the mammalian retina, the role of strong null-direction inhibition of On-Off direction-selective ganglion cells (On-Off DSGCs) on their direction selectivity is well-studied. However, how excitatory inputs influence the On-Off DSGC's visual response is underexplored. Here, we report that On-Off DSGCs have a spatially displaced glutamatergic receptive field along their horizontal preferred-null motion axes. This displaced receptive field contributes to DSGC null-direction spiking during interrupted motion trajectories. Theoretical analyses indicate that population responses during interrupted motion may help populations of On-Off DSGCs signal the spatial location of moving objects in complex, naturalistic visual environments. Our study highlights that the direction-selective circuit exploits separate sets of mechanisms under different stimulus conditions, and these mechanisms may help encode multiple visual features.

A high-density narrow-field inhibitory retinal interneuron with direct coupling to Müller glia.

Amacrine cells are interneurons composing the most diverse cell class in the mammalian retina. They help encode visual features such as edges or directed motion by mediating excitatory and inhibitory interactions between input (i.e. bipolar) and output (i.e. ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, metabolic regulation and neurovascular control. Here, we report that in mouse retina (of either sex), an abundant, though previously unstudied inhibitory amacrine cell is coupled directly to Müller glia. Electron microscopic reconstructions of this amacrine type revealed chemical synapses with known retinal cell types and extensive associations with Müller glia, the processes of which often completely ensheathe the neurites of this amacrine cell. Microinjecting small tracer molecules into the somas of these amacrine cells led to selective labelling of nearby Müller glia, leading us to suggest the name "Müller glia-coupled amacrine cell," or MAC. Our data also indicate that MACs release glycine at conventional chemical synapses, and viral retrograde transsynaptic tracing from the dorsal lateral geniculate nucleus (dLGN) showed selective connections between MACs and a subpopulation of RGC types. Visually-evoked responses revealed a strong preference for light increments; these "ON" responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling with other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function.Significance Statement:Gap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system and play multiple roles, including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells have rarely been reported and are poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Müller glia. Moreover, viral tracing, optogenetics and serial electron microscopy provide new information about the neuron's synaptic partners and physiological responses.

Local axonal morphology guides the topography of interneuron myelination in mouse and human neocortex.

GABAergic fast-spiking parvalbumin-positive (PV) interneurons are frequently myelinated in the cerebral cortex. However, the factors governing the topography of cortical interneuron myelination remain incompletely understood. Here, we report that segmental myelination along neocortical interneuron axons is strongly predicted by the joint combination of interbranch distance and local axon caliber. Enlargement of PV+ interneurons increased axonal myelination, while reduced cell size led to decreased myelination. Next, we considered regular-spiking SOM+ cells, which normally have relatively shorter interbranch distances and thinner axon diameters than PV+ cells, and are rarely myelinated. Consistent with the importance of axonal morphology for guiding interneuron myelination, enlargement of SOM+ cell size dramatically increased the frequency of myelinated axonal segments. Lastly, we confirm that these findings also extend to human neocortex by quantifying interneuron axonal myelination from ex vivo surgical tissue. Together, these findings establish a predictive model of neocortical GABAergic interneuron myelination determined by local axonal morphology.

Transcriptomic Signatures of Postnatal and Adult Intrinsically Photosensitive Ganglion Cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are rare mammalian photoreceptors essential for non-image-forming vision functions, such as circadian photoentrainment and the pupillary light reflex. They comprise multiple subtypes distinguishable by morphology, physiology, projections, and levels of expression of melanopsin (Opn4), their photopigment. The molecular programs that distinguish ipRGCs from other ganglion cells and ipRGC subtypes from one another remain elusive. Here, we present comprehensive gene expression profiles of early postnatal and adult mouse ipRGCs purified from two lines of reporter mice that mark different sets of ipRGC subtypes. We find dozens of novel genes highly enriched in ipRGCs. We reveal that Rasgrp1 and Tbx20 are selectively expressed in subsets of ipRGCs, though these molecularly defined groups imperfectly match established ipRGC subtypes. We demonstrate that the ipRGCs regulating circadian photoentrainment are diverse at the molecular level. Our findings reveal unexpected complexity in gene expression patterns across mammalian ipRGC subtypes.

µ-Opioid Receptor Activation Directly Modulates Intrinsically Photosensitive Retinal Ganglion Cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) encode light intensity and trigger reflexive responses to changes in environmental illumination. In addition to functioning as photoreceptors, ipRGCs are post-synaptic neurons in the inner retina, and there is increasing evidence that their output can be influenced by retinal neuromodulators. Here we show that opioids can modulate light-evoked ipRGC signaling, and we demonstrate that the M1, M2 and M3 types of ipRGCs are immunoreactive for µ-opioid receptors (MORs) in both mouse and rat. In the rat retina, application of the MOR-selective agonist DAMGO attenuated light-evoked firing ipRGCs in a dose-dependent manner (IC50 < 40 nM), and this effect was reversed or prevented by co-application of the MOR-selective antagonists CTOP or CTAP. Recordings from solitary ipRGCs, enzymatically dissociated from retinas obtained from melanopsin-driven fluorescent reporter mice, confirmed that DAMGO exerts its effect directly through MORs expressed by ipRGCs. Reduced ipRGC excitability occurred via modulation of voltage-gated potassium and calcium currents. These findings suggest a potential new role for endogenous opioids in the mammalian retina and identify a novel site of action-MORs on ipRGCs-through which opioids might exert effects on reflexive responses to environmental light.

Genetic access to neurons in the accessory optic system reveals a role for Sema6A in midbrain circuitry mediating motion perception.

The accessory optic system (AOS) detects retinal image slip and reports it to the oculomotor system for reflexive image stabilization. Here, we characterize two Cre lines that permit genetic access to AOS circuits responding to vertical motion. The first (Pcdh9-Cre) labels only one of the four subtypes of ON direction-selective retinal ganglion cells (ON-DS RGCs), those preferring ventral retinal motion. Their axons diverge from the optic tract just behind the chiasm and selectively innervate the medial terminal nucleus (MTN) of the AOS. Unlike most RGC subtypes examined, they survive after optic nerve crush. The second Cre-driver line (Pdzk1ip1-Cre) labels postsynaptic neurons in the MTN. These project predominantly to the other major terminal nucleus of the AOS, the nucleus of the optic tract (NOT). We find that the transmembrane protein semaphorin 6A (Sema6A) is required for the formation of axonal projections from the MTN to the NOT, just as it is for the retinal innervation of the MTN. These new tools permit manipulation of specific circuits in the AOS and show that Sema6A is required for establishing AOS connections in multiple locations.

The M6 cell: A small-field bistratified photosensitive retinal ganglion cell.

We have identified a novel, sixth type of intrinsically photosensitive retinal ganglion cell (ipRGC) in the mouse-the M6 cell. Its spiny, highly branched dendritic arbor is bistratified, with dendrites restricted to the inner and outer margins of the inner plexiform layer, co-stratifying with the processes of other ipRGC types. We show that M6 cells are by far the most abundant ganglion cell type labeled in adult pigmented Cdh3-GFP BAC transgenic mice. A few M5 ipRGCs are also labeled, but no other RGC types were encountered. Several distinct subnuclei in the geniculate complex and the pretectum contain labeled retinofugal axons in the Cdh3-GFP mouse. These are presumably the principle central targets of M6 cells (as well as M5 cells). Projections from M6 cells to the dorsal lateral geniculate nucleus were confirmed by retrograde tracing, suggesting they contribute to pattern vision. M6 cells have low levels of melanopsin expression and relatively weak melanopsin-dependent light responses. They also exhibit strong synaptically driven light responses. Their dendritic fields are the smallest and most abundantly branched of all ipRGCs. They have small receptive fields and strong antagonistic surrounds. Despite deploying dendrites partly in the OFF sublamina, M6 cells appear to be driven exclusively by the ON pathway, suggesting that their OFF arbor, like those of certain other ipRGCs, may receive ectopic input from passing ON bipolar cells axons in the OFF sublayer.

GABA release selectively regulates synapse development at distinct inputs on direction-selective retinal ganglion cells.

Synaptic inhibition controls a neuron's output via functionally distinct inputs at two subcellular compartments, the cell body and the dendrites. It is unclear whether the assembly of these distinct inhibitory inputs can be regulated independently by neurotransmission. In the mammalian retina, γ-aminobutyric acid (GABA) release from starburst amacrine cells (SACs) onto the dendrites of on-off direction-selective ganglion cells (ooDSGCs) is essential for directionally selective responses. We found that ooDSGCs also receive GABAergic input on their somata from other amacrine cells (ACs), including ACs containing the vasoactive intestinal peptide (VIP). When net GABAergic transmission is reduced, somatic, but not dendritic, GABAA receptor clusters on the ooDSGC increased in number and size. Correlative fluorescence imaging and serial electron microscopy revealed that these enlarged somatic receptor clusters are localized to synapses. By contrast, selectively blocking vesicular GABA release from either SACs or VIP ACs did not alter dendritic or somatic receptor distributions on the ooDSGCs, showing that neither SAC nor VIP AC GABA release alone is required for the development of inhibitory synapses in ooDSGCs. Furthermore, a reduction in net GABAergic transmission, but not a selective reduction from SACs, increased excitatory drive onto ooDSGCs. This increased excitation may drive a homeostatic increase in ooDSGC somatic GABAA receptors. Differential regulation of GABAA receptors on the ooDSGC's soma and dendrites could facilitate homeostatic control of the ooDSGC's output while enabling the assembly of the GABAergic connectivity underlying direction selectivity to be indifferent to altered transmission.

Light Affects Mood and Learning through Distinct Retina-Brain Pathways.

Light exerts a range of powerful biological effects beyond image vision, including mood and learning regulation. While the source of photic information affecting mood and cognitive functions is well established, viz. intrinsically photosensitive retinal ganglion cells (ipRGCs), the central mediators are unknown. Here, we reveal that the direct effects of light on learning and mood utilize distinct ipRGC output streams. ipRGCs that project to the suprachiasmatic nucleus (SCN) mediate the effects of light on learning, independently of the SCN's pacemaker function. Mood regulation by light, on the other hand, requires an SCN-independent pathway linking ipRGCs to a previously unrecognized thalamic region, termed perihabenular nucleus (PHb). The PHb is integrated in a distinctive circuitry with mood-regulating centers and is both necessary and sufficient for driving the effects of light on affective behavior. Together, these results provide new insights into the neural basis required for light to influence mood and learning.

The M5 Cell: A Color-Opponent Intrinsically Photosensitive Retinal Ganglion Cell.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) combine direct photosensitivity through melanopsin with synaptically mediated drive from classical photoreceptors through bipolar-cell input. Here, we sought to provide a fuller description of the least understood ipRGC type, the M5 cell, and discovered a distinctive functional characteristic-chromatic opponency (ultraviolet excitatory, green inhibitory). Serial electron microscopic reconstructions revealed that M5 cells receive selective UV-opsin drive from Type 9 cone bipolar cells but also mixed cone signals from bipolar Types 6, 7, and 8. Recordings suggest that both excitation and inhibition are driven by the ON channel and that chromatic opponency results from M-cone-driven surround inhibition mediated by wide-field spiking GABAergic amacrine cells. We show that M5 cells send axons to the dLGN and are thus positioned to provide chromatic signals to visual cortex. These findings underscore that melanopsin's influence extends beyond unconscious reflex functions to encompass cortical vision, perhaps including the perception of color.

A retinal code for motion along the gravitational and body axes.

Self-motion triggers complementary visual and vestibular reflexes supporting image-stabilization and balance. Translation through space produces one global pattern of retinal image motion (optic flow), rotation another. We examined the direction preferences of direction-sensitive ganglion cells (DSGCs) in flattened mouse retinas in vitro. Here we show that for each subtype of DSGC, direction preference varies topographically so as to align with specific translatory optic flow fields, creating a neural ensemble tuned for a specific direction of motion through space. Four cardinal translatory directions are represented, aligned with two axes of high adaptive relevance: the body and gravitational axes. One subtype maximizes its output when the mouse advances, others when it retreats, rises or falls. Two classes of DSGCs, namely, ON-DSGCs and ON-OFF-DSGCs, share the same spatial geometry but weight the four channels differently. Each subtype ensemble is also tuned for rotation. The relative activation of DSGC channels uniquely encodes every translation and rotation. Although retinal and vestibular systems both encode translatory and rotatory self-motion, their coordinate systems differ.

A subset of ipRGCs regulates both maturation of the circadian clock and segregation of retinogeniculate projections in mice.

The visual system consists of two major subsystems, image-forming circuits that drive conscious vision and non-image-forming circuits for behaviors such as circadian photoentrainment. While historically considered non-overlapping, recent evidence has uncovered crosstalk between these subsystems. Here, we investigated shared developmental mechanisms. We revealed an unprecedented role for light in the maturation of the circadian clock and discovered that intrinsically photosensitive retinal ganglion cells (ipRGCs) are critical for this refinement process. In addition, ipRGCs regulate retinal waves independent of light, and developmental ablation of a subset of ipRGCs disrupts eye-specific segregation of retinogeniculate projections. Specifically, a subset of ipRGCs, comprising ~200 cells and which project intraretinally and to circadian centers in the brain, are sufficient to mediate both of these developmental processes. Thus, this subset of ipRGCs constitute a shared node in the neural networks that mediate light-dependent maturation of the circadian clock and light-independent refinement of retinogeniculate projections.

A Cre Mouse Line for Probing Irradiance- and Direction-Encoding Retinal Networks.

Cell type-specific Cre driver lines have revolutionized the analysis of retinal cell types and circuits. We show that the transgenic mouse Rbp4-Cre selectively labels several retinal neuronal types relevant to the encoding of absolute light intensity (irradiance) and visual motion. In the ganglion cell layer (GCL), most marked cells are wide-field spiking polyaxonal amacrine cells (ACs) with sustained irradiance-encoding ON responses that persist during chemical synaptic blockade. Their arbors spread about 1 mm across the retina and are restricted to the inner half of the ON sublamina of the inner plexiform layer (IPL). There, they costratify with dendrites of M2 intrinsically photosensitive retinal ganglion cells (ipRGCs), to which they are tracer coupled. We propose that synaptically driven and intrinsic photocurrents of M2 cells pass through gap junctions to drive AC light responses. Also marked in this mouse are two types of RGCs. R-cells have a bistratified dendritic arbor, weak directional tuning, and irradiance-encoding ON responses. However, they also receive excitatory OFF input, revealed during ON-channel blockade. Serial blockface electron microscopic (SBEM) reconstruction confirms OFF bipolar input, and reveals that some OFF input derives from a novel type of OFF bipolar cell (BC). R-cells innervate specific layers of the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC). The other marked RGC type (RDS) is bistratified, transient, and ON-OFF direction selective (DS). It apparently innervates the nucleus of the optic tract (NOT). The Rbp4-Cre mouse will be valuable for targeting these cell types for further study and for selectively manipulating them for circuit analysis.

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells.

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2(-/-) mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2(-/-) were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2(-/-) mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light.