Visual Responses in Mice Lacking Critical Components of All Known Retinal Phototransduction Cascades

The mammalian visual system relies upon light detection by outer-retinal rod/cone photoreceptors and melanopsin-expressing retinal ganglion cells. Gnat1(-/-); Cnga3(-/-); Opn4(-/-) mice lack critical elements of each of these photoreceptive mechanisms via targeted disruption of genes encoding rod alpha transducin (Gnat1); the cone-specific alpha 3 cyclic nucleotide gated channel subunit (Cnga3); and melanopsin (Opn4). Although assumed blind, we show here that these mice retain sufficiently widespread retinal photoreception to drive a reproducible flash electroretinogram (ERG). The threshold sensitivity of this ERG is similar to that of cone-based responses, however it is lost under light adapted conditions. Its spectral efficiency is consistent with that of rod opsin, but not cone opsins or melanopsin, indicating that it originates with light absorption by the rod pigment. The TKO light response survives intravitreal injection of U73122 (a phospholipase C antagonist), but is inhibited by a missense mutation of cone alpha transducin (Gnat2(cpfl3)), suggesting Gnat2-dependence. Visual responses in TKO mice extend beyond the retina to encompass the lateral margins of the lateral geniculate nucleus and components of the visual cortex. Our data thus suggest that a Gnat1-independent phototransduction mechanism downstream of rod opsin can support relatively widespread responses in the mammalian visual system. This anomalous rod opsin-based vision should be considered in experiments relying upon Gnat1 knockout to silence rod phototransduction

Predicting anti-arthritic drug effects in collagen-induced arthritis using short-term mechanistic models of collagen II immunity

Novel anti-arthritic drugs are often assessed in murine collagen-induced arthritis (CIA), which is a widely used pre-clinical model of rheumatoid arthritis. However, CIA studies are lengthy, development of arthritis is not synchronised and not all animals develop disease. Work conducted in this thesis addressed some of these issues by developing short-term mechanistic models of collagen II (CII) immunity. Drug effects on CII-induced hypersensitivity, anti-CII antibodies and ex vivo CII stimulated CD4+ T cell proliferation in mice 14 days post-CII sensitisation were assessed and compared to their anti-arthritic effect in CIA. As this thesis progressed, it was reported that IL-17 secreting CD4+ T helper (Th17) cells represent a population of cells distinct from interferon gamma (IFNγ) secreting Th1 and IL-4 secreting Th2 cells and may be involved in autoimmune disease. However, the role of IL-17 and Th17 cells in CIA and CII immunity was not defined. The role of IL-17 and its relationship with Th1 and Th2 cells was investigated in the CII stimulated CD4+ T cell assay. Results showed that CII specific Th1 and Th17, but not Th2 cells, are present in cultures of cells from CII sensitised mice. Addition of anti-IL-17 to these cultures increased the number of CII specific IFNγ secreting CD4+ T cells. Literature evidence suggests that IFNγ is protective in CIA. This increase in IFNγ may therefore represent a novel mechanism of action by which anti-IL-17 exerts some of its anti-arthritic activity. This thesis has shown that short-term models of CII immunity can predict anti-arthritic drug effects in CIA. A novel screening cascade has been proposed which could be used in drug discovery and may reduce the number of animals required for CIA studies. Moreover, the differential effect of anti-arthritic drugs in these models suggests they can discriminate between drugs and identify novel mechanisms of action

Retrograde Melanopsin Signaling Increases With Age in Retinal Degenerate Mice Lacking Rods and the Majority of Cones

PURPOSE: Following on from reports of retrograde retinal signaling in mice, we sought to investigate the influence of age and retinal location on this phenomenon using mice that lack rods and the majority of cones. METHODS: We used functional anatomy for c-fos (Fos) and tyrosine hydroxylase (TH) to measure light-driven activation of dopamine neurons along a dorsal-ventral transect in C3H/He wild-type and rodless-coneless rd/rd cl (rdcl) mice aged 3, 5, and >14 months. A parallel series of retinae from 3-month-old mice was also stained for cone opsins and melanopsin. RESULTS: Analysis by confocal microscopy revealed light-driven Fos activation in TH cells residing in the middorsal retina of the youngest rdcl mice. This region was largely devoid of residual cones but contained a large number of intrinsically photosensitive retinal ganglion cells (ipRGCs) and the highest density of melanopsin neurites. With advancing age, there was a paradoxical increase in retrograde signaling from ∼3% Fos-positive (Fos+) TH cells at 3 months to ∼36% in rdcl mice >14 months. This increased activation occurred in more central and peripheral retinal regions. CONCLUSIONS: Our data provide new insights into the anatomy and plasticity of retrograde melanopsin signaling in mice with severe rod/cone dystrophy. The increased retrograde signaling we detect may result from either an increased potency of melanopsin signaling with advancing age and/or postsynaptic modification to dopaminergic neurons

Distribution of melanopsin positive neurons in pigmented and albino mice: evidence for melanopsin interneurons in the mouse retina.

Here we have studied the population of intrinsically photosensitive retinal ganglion cells (ipRGCs) in adult pigmented and albino mice. Our data show that although pigmented (C57Bl/6) and albino (Swiss) mice have a similar total number of ipRGCs, their distribution is slightly different: while in pigmented mice ipRGCs are more abundant in the temporal retina, in albinos the ipRGCs are more abundant in superior retina. In both strains, ipRGCs are located in the retinal periphery, in the areas of lower Brn3a(+)RGC density. Both strains also contain displaced ipRGCs (d-ipRGCs) in the inner nuclear layer (INL) that account for 14% of total ipRGCs in pigmented mice and 5% in albinos. Tracing from both superior colliculli shows that 98% (pigmented) and 97% (albino) of the total ipRGCs, become retrogradely labeled, while double immunodetection of melanopsin and Brn3a confirms that few ipRGCs express this transcription factor in mice. Rather surprisingly, application of a retrograde tracer to the optic nerve (ON) labels all ipRGCs, except for a sub-population of the d-ipRGCs (14% in pigmented and 28% in albino, respectively) and melanopsin positive cells residing in the ciliary marginal zone (CMZ) of the retina. In the CMZ, between 20% (pigmented) and 24% (albino) of the melanopsin positive cells are unlabeled by the tracer and we suggest that this may be because they fail to send an axon into the ON. As such, this study provides the first evidence for a population of melanopsin interneurons in the mammalian retina

A role for the ciliary marginal zone in the melanopsin-dependent intrinsic pupillary light reflex.

Maintenance of pupillary constriction in light-adapted rodents has traditionally been thought to involve a reflex between retina, brain and iris, with recent work identifying the melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) as the major conduits for retinal input to the brain. There is also a less well-understood phenomenon whereby the iris of some mammals, including mice, will constrict to light when either the eye, or the iris itself is physically isolated from the brain. The intrinsic pupillary light reflex (iPLR) is the term given to pupil constriction in the absence of retinal input to the brain. Here, using an intraocular axotomy approach, we show that the iPLR in conscious mice spans a dynamic range over 3 log units of irradiance. This iPLR response is absent in melanopsin knockout (MKO) mice and can be significantly inhibited by atropine. Immunohistochemistry for cfos and melanopsin, in combination with light exposure revealed a population of small ipRGCs in the retinal ciliary marginal zone (CMZ), which remain responsive to light in axotomised mice. We report that damage to the CMZ in a novel in vitro preparation removes a significant component of the iPLR response, while a detailed immunohistochemical analysis of the CMZ in wildtype mice revealed a melanopsin-rich plexus, which was consistently most intense in nasal retina. There were clear examples of melanopsin-positive, direct retino-ciliary projections, which appear to emanate from Brn3b negative, M1 type ipRGCs. These cells are clustered along the melanopsin-rich plexus nasally and may channel ipRGC signals from retina into the iris via ciliary body. Comparison between wildtype and MKO mice reveals that the ciliary body is also weakly stained for melanopsin. Our results show that the full extent of iPLR in mice requires cholinergic neurotransmission and intact signalling at the CMZ / ciliary body. This response may be mediated to some extent by ipRGCs, which send direct projections from the retina into ciliary body. In addition to the melanopsin-mediated iris sphincter constriction suggested by others, we propose a new mechanism, which may involve constriction of the ciliary body and ipRGC-mediated relaxation of the iris dilator muscle