Immunotoxin‐induced ablation of melanopsin retinal ganglion cells in a non‐murine mammalian model

In mammals, non‐image‐forming visual functions, including circadian photoentrainment and the pupillary light reflex, are thought to be mediated by the combination of rods, cones, and the melanopsin‐expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Although several genetic models have been developed to clarify the individual roles of the rod, cone, and ipRGC systems in mediating non‐image visual function, assessing the in vivo role(s) of the ipRGCs has been complicated by the possibility of ontogenetic issues in these genetically modified animal models. In the present study, we describe the development and validation of an immunotoxin that specifically targets the ipRGC population in the mature mammalian retina. This ipRGC immunotoxin, consisting of saporin conjugated to a melanopsin polyclonal antibody, was evaluated with respect to its effectiveness and specificity in depleting the ipRGC population in the fully developed rat retina. The results showed that the ipRGC toxin rapidly and permanently depleted ～70% of the ipRGC population, without inducing appreciable changes in the cell number or morphology of any of the non‐melanopsin‐containing retinal cell populations investigated. These findings suggest that the newly developed ipRGC immunotoxin provides a potent method for achieving relatively rapid, permanent, and selective depletion of the ipRGC population in a non‐murine model system. The development of this ipRGC‐ablation method is the next step in elucidating the role of ipRGCs in mediating non‐visual and circadian light‐resetting responses in a wide range of non‐murine mammalian models. J. Comp. Neurol. 516:125–140, 2009. © 2009 Wiley‐Liss, Inc.     

A retinal ganglion cell that can signal irradiance continuously for 10 hours

A recently discovered type of mammalian retinal ganglion cell encodes environmental light intensity and mediates non-image-forming visual behaviors, such as the pupillary reflex and circadian photoentrainment. These intrinsically photosensitive retinal ganglion cells (ipRGCs) generate endogenous, melanopsin-based photoresponses as well as extrinsic, rod/cone-driven responses. Because the ipRGCs' light responses and the behaviors they control are both remarkably tonic, these cells have been hypothesized to be capable of irradiance detection lasting throughout the day. I tested this hypothesis by obtaining multielectrode-array recordings from ipRGCs in a novel rat eyecup preparation that enhances the regeneration of rod/cone photopigments. I found that 10 h constant light could continuously evoke action potentials in these ganglion cells under conditions that stimulated (1) only melanopsin, (2) mainly the rod input, and (3) both intrinsic and extrinsic responses. In response to a 10 h stimulus with gradual intensity changes to simulate sunrise and sunset, ipRGC firing rates slowly increased during the "sunrise" phase and slowly decreased during the "sunset" phase. Furthermore, I recorded from putative ipRGCs of melanopsin-knock-out mice and found that these cells retained the ability to respond in a sustained fashion to 20 min light steps, indicating that melanopsin is not required for such tonic responses. In conclusion, ipRGCs can signal light continuously for at least 10 h and can probably track gradual irradiance changes over the course of the day. These results further suggest that the photoreceptors and ON bipolar cells presynaptic to ipRGCs may be able to respond to light continuously for 10 h.     

Two types of melanopsin retinal ganglion cell differentially innervate the hypothalamic suprachiasmatic nucleus and the olivary pretectal nucleus

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) innervate the hypothalamic suprachiasmatic nucleus (SCN) and the olivary pretectal nucleus (OPN), providing irradiance information for entrainment of circadian rhythms and for stimulating the pupillary light reflex. In this study, mice were used in which the melanopsin gene was replaced with the tau-lacZ gene. Heterozygous ( tau-lacZ ^+/– ) mice express both melanopsin and β-galactosidase. In tau-lacZ ^+/– mice, only ∼50% of melanopsin ipRGCs contain β-galactosidase, and these cells are specifically labeled with a C-terminus melanopsin antibody. Retrograde tracer injection into the SCN labels β-galactosidase-expressing ipRGCs (termed M1) that comprise ∼80% of the SCN-projecting ipRGCs. M1 ipRGCs and an additional set of ipRGCs (termed M2) are labeled with a melanopsin antiserum targeted against the N-terminus of the melanopsin protein; M2 ipRGCs do not contain detectable β-galactosidase, and these cells make up the remainder of the SCN-projecting RGCs. Tracer injection into the OPN labeled non-melanopsin RGCs and both types of melanopsin ipRGC: 45% M1 and 55% M2. Infection of the iris with pseudorabies virus (PRV) results in retrograde transneuronal label of OPN projection neurons that innervate preganglionic parasympathetic neurons of the Edinger-Westphal nucleus; PRV-labeled cells were located almost exclusively within the terminal field of M1 ipRGCs in the periphery (shell) of the OPN. The OPN core receives retinal input, and we hypothesize that the OPN core receives input from the M2 ipRGCs. Two subtypes of melanopsin ipRGCs project differentially to the SCN and OPN; the functional significance of ipRGCs subtypes is currently unknown.     

Intrinsically photosensitive retinal ganglion cells: many subtypes, diverse functions

For decades, rods and cones were thought to be the only photoreceptors in the mammalian retina. However, a population of atypical photoreceptive retinal ganglion cells (RGCs) expresses the photopigment melanopsin and is intrinsically photosensitive (ipRGCs). These ipRGCs are crucial for relaying light information from the retina to the brain to control circadian photoentrainment, pupillary light reflex, and sleep. ipRGCs were initially described as a uniform population involved solely in signaling irradiance for non-image forming functions. Recent work, however, has uncovered that ipRGCs are unexpectedly diverse at the molecular, cellular and functional levels, and could even be involved in image formation. This review summarizes our current understanding of the diversity of ipRGCs and their various roles in modulating behavior.     

Mice with early retinal degeneration show differences in neuropeptide expression in the suprachiasmatic nucleus

Background  

In mammals, the brain clock responsible for generating circadian rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light entrainment of the clock occurs through intrinsically photosensitive retinal ganglion cells (ipRGCs) whose axons project to the SCN via the retinohypothalamic tract. Although ipRGCs are sufficient for photoentrainment, rod and cone photoreceptors also contribute. Adult CBA/J mice, which exhibit loss of rod and cone photoreceptors during early postnatal development, have greater numbers of ipRGCs compared to CBA/N control mice. A greater number of photosensitive cells might argue for enhanced light responses, however, these mice exhibit attenuated phase shifting behaviors. To reconcile these findings, we looked for potential differences in SCN neurons of CBA/J mice that might underly the altered circadian behaviors. We hypothesized that CBA/J mice have differences in the expression of neuropeptides in the SCN, where ipRGCs synapse. The neuropeptides vasoactive intestinal peptide (VIP) and vasopressin (VP) are expressed by many SCN neurons and play an important role in the generation of circadian rhythms and photic entrainment.     

Distribution and diversity of intrinsically photosensitive retinal ganglion cells in tree shrew

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate the pupillary light reflex, circadian entrainment, and may contribute to luminance and color perception. The diversity of ipRGCs varies from rodents to primates, suggesting differences in their contributions to retinal output. To further understand the variability in their organization and diversity across species, we used immunohistochemical methods to examine ipRGCs in tree shrew (Tupaia belangeri). Tree shrews share membership in the same clade, or evolutionary branch, as rodents and primates. They are highly visual, diurnal animals with a cone-dominated retina and a geniculo-cortical organization resembling that of primates. We identified cells with morphological similarities to M1 and M2 cells described previously in rodents and primates. M1-like cells typically had somas in the ganglion cell layer, with 23% displaced to the inner nuclear layer (INL). However, unlike M1 cells, they had bistratified dendritic fields ramifying in S1 and S5 that collectively tiled space. M2-like cells had dendritic fields restricted to S5 that were smaller and more densely branching. A novel third type of melanopsin immunopositive cell was identified. These cells had somata exclusively in the INL and monostratified dendritic fields restricted to S1 that tiled space. Surprisingly, these cells immunolabeled for tyrosine hydroxylase, a key component in dopamine synthesis. These cells immunolabeled for an RGC marker, not amacrine cell markers, suggesting that they are dopaminergic ipRGCs. We found no evidence for M4 or M5 ipRGCs, described previously in rodents. These results identify some organizational features of the ipRGC system that are canonical versus species-specific.     

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.     

Intrinsic phototransduction persists in melanopsin-expressing ganglion cells lacking diacylglycerol-sensitive TRPC subunits

In mammals, intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate various non-image-forming photic responses, such as circadian photoentrainment, pupillary light reflex and pineal melatonin suppression. ipRGCs directly respond to environmental light by activation of the photopigment melanopsin followed by the opening of an unidentified cation-selective channel. Studies in heterologous expression systems and in the native retina have strongly implicated diacylglycerol-sensitive transient receptor potential channels containing TRPC3, TRPC6 and TRPC7 subunits in melanopsin-evoked depolarization. Here we show that melanopsin-evoked electrical responses largely persist in ipRGCs recorded from early postnatal (P6-P8) and adult (P22-P50) mice lacking expression of functional TRPC3, TRPC6 or TRPC7 subunits. Multielectrode array (MEA) recordings performed at P6-P8 stages under conditions that prevent influences from rod/cone photoreceptors show comparable light sensitivity for the melanopsin-evoked responses in these mutant mouse lines in comparison to wild-type (WT) mice. Patch-clamp recordings from adult mouse ipRGCs lacking TRPC3 or TRPC7 subunits show intrinsic light-evoked responses equivalent to those recorded in WT mice. Persistence of intrinsic light-evoked responses was also noted in ipRGCs lacking TRPC6 subunits, although with significantly smaller magnitudes. These results demonstrate that the melanopsin-evoked depolarization in ipRGCs is not mediated by either TRPC3, TRPC6 or TRPC7 channel subunits alone. They also suggest that the melanopsin signaling pathway includes TRPC6-containing heteromeric channels in mature retinas.     

Photoreceptive Ganglion Cells Drive Circuits for Local Inhibition in the Mouse Retina

Intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit melanopsin-dependent light responses that persist in the absence of rod and cone photoreceptor-mediated input. In addition to signaling anterogradely to the brain, ipRGCs signal retrogradely to intraretinal circuitry via gap junction-mediated electrical synapses with amacrine cells (ACs). However, the targets and functions of these intraretinal signals remain largely unknown. Here, in mice of both sexes, we identify circuitry that enables M5 ipRGCs to locally inhibit retinal neurons via electrical synapses with a nonspiking GABAergic AC. During pharmacological blockade of rod- and cone-mediated input, whole-cell recordings of corticotropin-releasing hormone-expressing (CRH⁺) ACs reveal persistent visual responses that require both melanopsin expression and gap junctions. In the developing retina, ipRGC-mediated input to CRH⁺ ACs is weak or absent before eye opening, indicating a primary role for this input in the mature retina (i.e., in parallel with rod- and cone-mediated input). Among several ipRGC types, only M5 ipRGCs exhibit consistent anatomical and physiological coupling to CRH⁺ ACs. Optogenetic stimulation of local CRH⁺ ACs directly drives IPSCs in M4 and M5, but not M1-M3, ipRGCs. CRH⁺ ACs also inhibit M2 ipRGC-coupled spiking ACs, demonstrating direct interaction between discrete networks of ipRGC-coupled interneurons. Together, these results demonstrate a functional role for electrical synapses in translating ipRGC activity into feedforward and feedback inhibition of local retinal circuits.SIGNIFICANCE STATEMENT Melanopsin directly generates light responses in intrinsically photosensitive retinal ganglion cells (ipRGCs). Through gap junction-mediated electrical synapses with retinal interneurons, these uniquely photoreceptive RGCs may also influence the activity and output of neuronal circuits within the retina. Here, we identified and studied an electrical synaptic circuit that, in principle, could couple ipRGC activity to the chemical output of an identified retinal interneuron. Specifically, we found that M5 ipRGCs form electrical synapses with corticotropin-releasing hormone-expressing amacrine cells, which locally release GABA to inhibit specific RGC types. Thus, ipRGCs are poised to influence the output of diverse retinal circuits via electrical synapses with interneurons.     

Synaptic inputs to retinal ganglion cells that set the circadian clock

Melanopsin-containing retinal ganglion cells (RGCs) project to the suprachiasmatic nuclei (SCN) and mediate photoentrainment of the circadian system. Melanopsin is a novel retinal-based photopigment that renders these cells intrinsically photosensitive (ip). Although genetic ablation of melanopsin abolishes the intrinsic light response, it has a surprisingly minor effect on circadian photoentrainment. This and other non-visual responses to light are lost only when the melanopsin deficiency is coupled with mutations that disable classical rod and cone photoreceptors, suggesting that melanopsin-containing RGCs also receive rod- and cone-driven synaptic inputs. Using whole-cell patch-clamp recording, we demonstrate that light triggers synaptic currents in ipRGCs via activation of ionotropic glutamate and gamma-aminobutyric acid (GABA) receptors. Miniature postsynaptic currents (mPSCs) were clearly observed in ipRGCs, although they were less robust and were seen less frequently than those seen in non-ip cells. Pharmacological treatments revealed that the majority of ipRGCs receive excitatory glutamatergic inputs that were blocked by DNQX and/or kynurenic acid, as well as inhibitory GABAergic inputs that were blocked by bicuculline. Other ipRGCs received either glutamatergic or GABAergic inputs nearly exclusively. Although strychnine (Strych)-sensitive mPSCs were evident on many non-ipRGCs, indicating the presence of glycinergic inputs, we saw no evidence of Strych-sensitive events in ipRGCs. Based on these results, it is clear that SCN-projecting RGCs can respond to light both via an intrinsic melanopsin-based signaling cascade and via a synaptic pathway driven by classical rod and/or cone photoreceptors. It remains to be determined how the ipRGCs integrate these temporally distinct inputs to generate the signals that mediate circadian photoentrainment and other non-visual responses to light.     

The development of melanopsin-containing retinal ganglion cells in mice with early retinal degeneration

In mammals, the neuronal pathways by which rod and cone photoreceptors mediate vision have been well documented. The roles that classical photoreceptors play in photoentrainment, however, have been less clear. In mammals, intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the photopigment melanopsin project directly to the suprachiasmatic nucleus of the hypothalamus, the site of the circadian clock, and thereby contribute to non-image-forming responses to light. Classical photoreceptors are not necessary for photoentrainment as loss of rods and cones does not eliminate light entrainment. Conflicting evidence arose, however, when attenuated phase-shifting responses were observed in the retinal-degenerate CBA/J mouse. In this study, we examined the time course of retinal degeneration in CBA/J mice and used these animals to determine if maturation of the outer retina regulates the morphology, number and distribution of ipRGCs. We also examined whether degeneration during the early development of the outer retina can alter the function of the adult circadian system. We report that dendritic stratification and distribution of ipRGCs was unaltered in mice with early retinal degeneration, suggesting that normal development of the outer retina was not necessary for these processes. We found, however, that adult CBA/J mice have greater numbers of ipRGCs than controls, implicating a role for the outer retinal photoreceptors in regulating developmental cell death of ipRGCs.     

Melanopsin expressing human retinal ganglion cells: Subtypes,distribution, and intraretinal connectivity

Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex, and sleep/wake cycles. The different functions seem to be associated to different subtypes of melanopsin cells. In rodents, subtype classification has associated subtypes to function. In primate and human retina such classification has so far, not been applied. In the present study using antibodies against N‐ and C‐terminal parts of human melanopsin, confocal microscopy and 3D reconstruction of melanopsin immunoreactive (‐ir) RGCs, we applied the criteria used in mouse on human melanopsin‐ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin‐ir RGCs, which were named “gigantic M1 (GM1)” and “gigantic displaced M1 (GDM1).” Few M3 cells and no M5 subtypes were labeled. Total cell counts from one male and one female retina revealed that the human retina contains 7283 ± 237 melanopsin‐ir (0.63–0.75% of the total number of RGCs). The melanopsin subtypes were unevenly distributed. Most significant was the highest density of M4 cells in the nasal retina. We identified input to the melanopsin‐ir RGCs from AII amacrine cells and directly from rod bipolar cells via ribbon synapses in the innermost ON layer of the inner plexiform layer (IPL) and from dopaminergic amacrine cells and GABAergic processes in the outermost OFF layer of the IPL. The study characterizes a heterogenic population of human melanopsin‐ir RGCs, which most likely are involved in different functions.     

A Role for Thalamic Projection GABAergic Neurons in Circadian Responses to Light

The thalamus is an important hub for sensory information and participates in sensory perception, regulation of attention, arousal and sleep. These functions are executed primarily by glutamatergic thalamocortical neurons that extend axons to the cortex and initiate cortico-thalamocortical connectional loops. However, the thalamus also contains projection GABAergic neurons that do not extend axons toward the cortex. Here, we have harnessed recent insight into the development of the intergeniculate leaflet (IGL) and the ventral lateral geniculate nucleus (LGv) to specifically target and manipulate thalamic projection GABAergic neurons in female and male mice. Our results show that thalamic GABAergic neurons of the IGL and LGv receive retinal input from diverse classes of retinal ganglion cells (RGCs) but not from the M1 intrinsically photosensitive retinal ganglion cell (ipRGC) type. We describe the synergistic role of the photoreceptor melanopsin and the thalamic neurons of the IGL/LGv in circadian entrainment to dim light. We identify a requirement for the thalamic IGL/LGv neurons in the rapid changes in vigilance states associated with circadian light transitions.SIGNIFICANCE STATEMENT The intergeniculate leaflet (IGL) and ventral lateral geniculate nucleus (LGv) are part of the extended circadian system and mediate some nonimage-forming visual functions. Here, we show that each of these structures has a thalamic (dorsal) as well as prethalamic (ventral) developmental origin. We map the retinal input to thalamus-derived cells in the IGL/LGv complex and discover that while RGC input is dominant, this is not likely to originate from M1ipRGCs. We implicate thalamic cells in the IGL/LGv in vigilance state transitions at circadian light changes and in overt behavioral entrainment to dim light, the latter exacerbated by concomitant loss of melanopsin expression.     

Crosstalk: The diversity of melanopsin ganglion cell types has begun to challenge the canonical divide between image-forming and non-image-forming vision

Melanopsin ganglion cells have defied convention since their discovery almost 20 years ago. In the years following, many types of these intrinsically photosensitive retinal ganglion cells (ipRGCs) have emerged. In the mouse retina, there are currently six known types (M1–M6) of melanopsin ganglion cells, each with unique morphology, mosaics, connections, physiology, projections, and functions. While melanopsin-expressing cells are usually associated with behaviors like circadian photoentrainment and the pupillary light reflex, the characterization of multiple types has demonstrated a reach that may extend far beyond non-image-forming vision. In fact, studies have shown that individual types of melanopsin ganglion cells have the potential to impact image-forming functions like contrast sensitivity and color opponency. Thus, the goal of this review is to summarize the morphological and functional aspects of the six known types of melanopsin ganglion cells in the mouse retina and to highlight their respective roles in non-image-forming and image-forming vision. Although many melanopsin ganglion cell types do project to image-forming brain targets, it is important to note that this is only the first step in determining their influence on image-forming vision. Even so, the visual system has canonically been divided into these two functional realms and melanopsin ganglion cells have begun to challenge the boundary between them, providing an overlap of visual information that is complementary rather than redundant. Further studies on these ganglion cell photoreceptors will no doubt continue to illustrate an ever-expanding role for melanopsin ganglion cells in image-forming vision.     

Melanopsin-positive intrinsically photosensitive retinal ganglion cells: from form to function

Melanopsin imparts an intrinsic photosensitivity to a subclass of retinal ganglion cells (ipRGCs). Generally thought of as irradiance detectors, ipRGCs target numerous brain regions involved in non-image-forming vision. ipRGCs integrate their intrinsic, melanopsin-mediated light information with rod/cone signals relayed via synaptic connections to influence light-dependent behaviors. Early observations indicated diversity among these cells and recently several specific subtypes have been identified. These subtypes differ in morphological and physiological form, controlling separate functions that range from biological rhythm via circadian photoentrainment, to protective behavioral responses including pupil constriction and light avoidance, and even image-forming vision. In this Mini-Symposium review, we will discuss some recent findings that highlight the diversity in both form and function of these recently discovered atypical photoreceptors.     

Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are entrained to the environmental light/dark cycle via intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin and the neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP). The ipRGCs regulate other nonimage‐forming visual functions such as the pupillary light reflex, masking behavior, and light‐induced melatonin suppression. To evaluate whether PACAP‐immunoreactive retinal projections are useful as a marker for central projection of ipRGCs in the monkey brain, we characterized the occurrence of PACAP in melanopsin‐expressing ipRGCs and in the retinal target areas in the brain visualized by the anterograde tracer cholera toxin subunit B (CtB) in combination with PACAP staining. In the retina, PACAP and melanopsin were found to be costored in 99% of melanopsin‐expressing cells characterized as inner and outer stratifying melanopsin RGCs. Two macaque monkeys were anesthetized and received a unilateral intravitreal injection of CtB. Bilateral retinal projections containing colocalized CtB and PACAP immunostaining were identified in the SCN, the lateral geniculate complex including the pregeniculate nucleus, the pretectal olivary nucleus, the nucleus of the optic tract, the brachium of the superior colliculus, and the superior colliculus. In conclusion, PACAP‐immunoreactive projections with colocalized CtB represent retinal projections of ipRGCs in the macaque monkey, supporting previous retrograde tracer studies demonstrating that melanopsin‐containing retinal projections reach areas in the primate brain involved in both image‐ and nonimage‐forming visual processing. J. Comp. Neurol. 522:2231–2248, 2014. © 2014 Wiley Periodicals, Inc.