Hsi Wen Liao

Melanopsin-expressing ganglion cells in primate retina signal colour and irradiance and project to the LGN

Human vision starts with the activation of rod photoreceptors in dim light and short (S)-, medium (M)-, and long (L)- wavelength-sensitive cone photoreceptors in daylight. Recently a parallel, non-rod, non-cone photoreceptive pathway, arising from a population of retinal ganglion cells, was discovered in nocturnal rodents. These ganglion cells express the putative photopigment melanopsin and by signalling gross changes in light intensity serve the subconscious, ‘non-image-forming’ functions of circadian photoentrainment and pupil constriction. Here we show an anatomically distinct population of ‘giant’, melanopsin-expressing ganglion cells in the primate retina that, in addition to being intrinsically photosensitive, are strongly activated by rods and cones, and display a rare, S-Off, (L + M)-On type of colour-opponent receptive field. The intrinsic, rod and (L + M) cone-derived light responses combine in these giant cells to signal irradiance over the full dynamic range of human vision. In accordance with cone-based colour opponency, the giant cells project to the lateral geniculate nucleus, the thalamic relay to primary visual cortex. Thus, in the diurnal trichromatic primate, ‘non-image-forming’ and conventional ‘image-forming’ retinal pathways are merged, and the melanopsin-based signal might contribute to conscious visual perception.

Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision

Rod and cone photoreceptors detect light and relay this information through a multisynaptic pathway to the brain by means of retinal ganglion cells (RGCs). These retinal outputs support not only pattern vision but also non-image-forming (NIF) functions, which include circadian photoentrainment and pupillary light reflex (PLR). In mammals, NIF functions are mediated by rods, cones and the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs). Rod–cone photoreceptors and ipRGCs are complementary in signalling light intensity for NIF functions. The ipRGCs, in addition to being directly photosensitive, also receive synaptic input from rod–cone networks. To determine how the ipRGCs relay rod–cone light information for both image-forming and non-image-forming functions, we genetically ablated ipRGCs in mice. Here we show that animals lacking ipRGCs retain pattern vision but have deficits in both PLR and circadian photoentrainment that are more extensive than those observed in melanopsin knockouts. The defects in PLR and photoentrainment resemble those observed in animals that lack phototransduction in all three photoreceptor classes. These results indicate that light signals for irradiance detection are dissociated from pattern vision at the retinal ganglion cell level, and animals that cannot detect light for NIF functions are still capable of image formation.

Photon capture and signalling by melanopsin retinal ganglion cells

A subset of retinal ganglion cells has recently been discovered to be intrinsically photosensitive, with melanopsin as the pigment. These cells project primarily to brain centres for non-image-forming visual functions such as the pupillary light reflex and circadian photoentrainment. How well they signal intrinsic light absorption to drive behaviour remains unclear. Here we report fundamental parameters governing their intrinsic light responses and associated spike generation. The membrane density of melanopsin is 104-fold lower than that of rod and cone pigments, resulting in a very low photon catch and a phototransducing role only in relatively bright light. Nonetheless, each captured photon elicits a large and extraordinarily prolonged response, with a unique shape among known photoreceptors. Notably, like rods, these cells are capable of signalling single-photon absorption. A flash causing a few hundred isomerized melanopsin molecules in a retina is sufficient for reaching threshold for the pupillary light reflex.

Parietal-Eye Phototransduction Components and Their Potential Evolutionary Implications

The parietal-eye photoreceptor is unique because it has two antagonistic light signaling pathways in the same cell—a hyperpolarizing pathway maximally sensitive to blue light and a depolarizing pathway maximally sensitive to green light. Here, we report the molecular components of these two pathways. We found two opsins in the same cell: the blue-sensitive pinopsin and a previously unidentified green-sensitive opsin, which we name parietopsin. Signaling components included gustducin-α and Gαo, but not rod or cone transducin-α. Single-cell recordings demonstrated that Go mediates the depolarizing response. Gustducin-α resembles transducin-α functionally and likely mediates the hyperpolarizing response. The parietopsin-Go signaling pair provides clues about how rod and cone phototransduction might have evolved.

Non-image-forming ocular photoreception in vertebrates

It has been accepted for a hundred years or more that rods and cones are the only photoreceptive cells in the retina. The light signals generated in rods and cones, after processing by downstream retinal neurons (bipolar, horizontal, amacrine and ganglion cells), are transmitted to the brain via the axons of the ganglion cells for further analysis. In the past few years, however, convincing evidence has rapidly emerged indicating that a small subset of retinal ganglion cells in mammals is also intrinsically photosensitive. Melanopsin is the signaling photopigment in these cells. The main function of the inner-retina photoreceptors is to generate and transmit non-image-forming visual information, although some role in conventional vision (image detection) is also possible.

Melanopsin‐expressing ganglion cells on macaque and human retinas form two morphologically distinct populations

The long‐term goal of this research is to understand how retinal ganglion cells that express the photopigment melanopsin, also known as OPN4, contribute to vision in humans and other primates. Here we report the results of anatomical studies using our polyclonal antibody specifically against human melanopsin that confirm and extend previous descriptions of melanopsin cells in primates. In macaque and human retina, two distinct populations of melanopsin cells were identified based on dendritic stratification in either the inner or the outer portion of the inner plexiform layer (IPL). Variation in dendritic field size and cell density with eccentricity was confirmed, and dendritic spines, a new feature of melanopsin cells, were described. The spines were the sites of input from DB6 diffuse bipolar cell axon terminals to the inner stratifying type of melanopsin cells. The outer stratifying melanopsin type received inputs from DB6 bipolar cells via a sparse outer axonal arbor. Outer stratifying melanopsin cells also received inputs from axon terminals of dopaminergic amacrine cells. On the outer stratifying melanopsin cells, ribbon synapses from bipolar cells and conventional synapses from amacrine cells were identified in electron microscopic immunolabeling experiments. Both inner and outer stratifying melanopsin cell types were retrogradely labeled following tracer injection in the lateral geniculate nucleus (LGN). In addition, a method for targeting melanopsin cells for intracellular injection using their intrinsic fluorescence was developed. This technique was used to demonstrate that melanopsin cells were tracer coupled to amacrine cells and would be applicable to electrophysiological experiments in the future. J. Comp. Neurol. 524:2845–2872, 2016.

In vivo gene delivery in the retina using polyethylenimine

Virus-based methods are widely used in the mammalian nervous system for expressing genes (1) and for producing short hairpin RNAs (shRNAs) (2) to knock down genes by RNA interference (RNAi). In the latter case, only a percentage of the small interfering RNAs (siRNAs) can effectively silence their cognate target genes (3). Thus, multiple selections of siRNA are often required. This multiplicity can make the virus-based method time-, labor-, or cost-intensive, especially when compounded with the goal of targeting multiple genes. Also, not all neuronal types are susceptible targets of a viral carrier. For example, retinal ganglion cells (RGCs) are known so far to be readily transduced only by adeno-associated virus and lentiviruses (4–6). Here, we report the successful use of a polymer as a carrier to deliver shRNA-expressing plasmid DNA to these cells in vivo. Nonviral carriers such as polymers are simple to use and can be safer than viral carriers. The cationic polymer polyethylenimine (PEI) has been used for transfection in vitro and in vivo mostly of non-neuronal tissues (7). We tested the efficiency of PEI/DNA polyplexes for transfecting RGCs in vivo, so chosen because these cells are adjacent to the vitreous and therefore are likely accessible to the polyplexes delivered by intravitreal injection. A commercial vector (RNAi-Ready pSIREN-DNR-DsRed-Express; Clontech, Mountain View, CA, USA) expressing shRNA (driven by a human U6 promoter) and a reporter Discosoma red fluorescent protein (DsRed) [driven by a cytomegalovirus (CMV) promoter] was mixed with PEI (in vivo-jetPEI™; Polyplus Transfection, Illkirch, France) according to the manufacturer’s instructions. In this study, the N:P ratio (i.e., the number of nitrogen residues of in vivo-jetPEI per DNA phosphate) used was 10 (e.g., 1 μg DNA was mixed with 0.2 μL in vivo-jetPEI). The PEI/DNA polyplex solution (1.2 μL for each eye) was carefully administered intravitreally (Figure 1A) from the posterior-temporal side of the eye of an anesthetized mouse via a no. 33 custom needle (1-inch-long/sharp point/type no. 2; Hamilton, Reno, NV, USA) on a 2.5-μL Hamilton syringe. The resulting expression of DsRed was evident in many cells in the ganglion cell layer (Figure 1B). In the retinal cross-section, DsRed expression was confirmed to be in the ganglion cell layer, indicating that the intravitreally injected polyplexes were able to cross the optic nerve fiber layer (containing the axons of the ganglion cells) from the vitreous (Figure 1C). Figure 1 In vivo polymer-mediated gene delivery in retina We next examined the shRNA expression. We designed the shRNA to target melanopsin (8), the photopigment mediating the light response of the intrinsically photosensitive RGCs (ipRGCs) (9,10). The intrinsic photosensitivity of the ipRGCs is required in order for the pupillary light reflex to reach completion at high irradiances, with melanopsin-knockout mice showing an incomplete pupil restriction in bright light (9). We hypothesized that knocking down melanopsin in the wild-type mouse retina should produce a similar phenotype. We chose an albino background (Balb/c) because the pupil size of melanopsin-knockout mouse with this background (B6.129.Balb/c) at high irradiances was significantly larger than that with a pigmented background (B6.129; unpublished observation). The underlying mechanism for this difference is unclear, but may reflect multiple defects associated with the albino locus, including abnormal axonal projections from the eye to the brain, an underdeveloped central retina, and a deficit of the rod system (11). The pupil reflex in an albino background thus gave a more dramatic indication of melanopsin knockdown. In Figure 2A, immunohistochemistry with a melanopsin antibody (12) indicated that melanopsin expression in the transfected (DsRed positive) area was reduced to an undetectable level by the melanopsin-specific shRNA. In the untransfected (DsRed negative) area of the same retina, melanopsin expression was normal. The eyes injected with melanopsin-shRNA-expressing plasmid DNA showed an incomplete pupil constriction in bright light (10,000 lux), whereas the eyes injected with control DNA were unaffected (Figure 2, B and C). The variation in pupil constriction from eye to eye due to melanopsin-specific shRNA (Figure 2C) conceivably resulted from the variation in the size of the transfected area. Over the entire retina, the average number of remaining melanopsin-immunoreactive cells (all found outside the transfected area) at 5 days after transfection was 176 ± 28 (mean ± SEM; 12 retinas from 6 animals), which translated to 25% ± 4% of the total melanopsin-espressing RGCs (MOP-RGCs) (12). Thus, the average transfected area was 75% ± 4% (mean ± SEM). The variation in the transfected area presumably reflects the technically challenging intravitreal injection into the very limited space between the retina and the lens in mouse. In other mammals, including primates, the intra-vitreal injection should be considerably easier. Part of the variation in pupil size, which has autonomic input from the nervous system (9), could also have come from the animal’s stress level during handling (13). Nonetheless, the melanopsin-knockdown effect was clear. The DsRed expression and the melanopsin-knockdown effect started to appear as early as 16 h after injection and lasted at least 2 months (Figure 2D). Figure 2 Melanopsin short hairpin RNA (shRNA) abolished melanopsin expression in transfected area of retina and decreased pupillary light reflex at high irradiances We thus have demonstrated a fast and simple nonviral method using a polymer for delivering DNA to RGCs. From sequence design to injection, it can be as fast as a few days in the case of an RNAi experiment involving even multiple constructs. Recently, it has been shown possible to restore retinal photosensitivity in mice that have degenerated rods and cones by the virus-mediated expression of channelrhodopsin, a photosensitive ion channel, in RGCs (14). Thus, there is considerable research interest in vision reviving gene therapy involving these cells. The current method provides a simple alternative approach. Conceivably, with injection into the subretinal space, the same method can be used for gene delivery to the rods and cones, another active area of research on ameliorating loss of vision associated with defects in rod/cone function (15). As mentioned earlier, the polymer method becomes particularly expedient when the deliveries of many DNA constructs have to be tested or made.