[Show abstract][Hide abstract] ABSTRACT: Photoreception in the mammalian retina is not restricted to rods and cones but extends to a subset of retinal ganglion cells expressing the photopigment melanopsin (mRGCs). These mRGCs are known to drive such reflex light responses as circadian photoentrainment and pupillomotor movements. By contrast, until now there has been no direct assessment of their contribution to conventional visual pathways. Here, we address this deficit. Using new reporter lines, we show that mRGC projections are much more extensive than previously thought and extend across the dorsal lateral geniculate nucleus (dLGN), origin of thalamo-cortical projection neurons. We continue to show that this input supports extensive physiological light responses in the dLGN and visual cortex in mice lacking rods+cones (a model of advanced retinal degeneration). Moreover, using chromatic stimuli to isolate melanopsin-derived responses in mice with an intact visual system, we reveal strong melanopsin input to the ∼40% of neurons in the LGN that show sustained activation to a light step. We demonstrate that this melanopsin input supports irradiance-dependent increases in the firing rate of these neurons. The implication that melanopsin is required to accurately encode stimulus irradiance is confirmed using melanopsin knockout mice. Our data establish melanopsin-based photoreception as a significant source of sensory input to the thalamo-cortical visual system, providing unique irradiance information and allowing visual responses to be retained even in the absence of rods+cones. These findings identify mRGCs as a potential origin for aspects of visual perception and indicate that they may support vision in people suffering retinal degeneration.
[Show abstract][Hide abstract] ABSTRACT: Sustained responses are deficient in melanopsin knockout LGN neurons. Average (± SEM) response of all Opn4−/− cells (n = 217) to 460 nm light compared with responses of all Opn1mwR neurons (i.e. “sustained” + “transient” subpopulations, n = 248) to 460 nm (top) and 655 nm (bottom). Responses of Opn4−/− neurons were significantly smaller than those of Opn1mwR cells at 460 nm but similar to those at 655 nm (mean ± SEM increases over baseline 0–60 s after light on; 1.3±0.3, 3.3±0.5, and 1.1±0.3 spikes/s, respectively; one-way ANOVA with Bonferroni post-test, p<0.001 and p>0.05, respectively).
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[Show abstract][Hide abstract] ABSTRACT: Light response waveforms in thalamic sub-regions of rd/rd cl mice. The average single unit response waveforms following 60 s, 460 nm, illumination in rd/rd cl mice were similar regardless of the projected anatomical location of the cell. Data show the mean ± SEM change in firing rate of light responsive cells detected in the dorsal LGN (top; n = 84), intergeniculate leaflet (IGL) and ventral LGN (middle; n = 221), or medial areas of the thalamus bordering the LGN (bottom; n = 39).
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[Show abstract][Hide abstract] ABSTRACT: Comparative anatomy of monocular retinal projections in rd/rd cl and Opn1mwR mice. Representative sections (100 µm thick) from rd/rd cl (left) and Opn1mwR (right) mice showing cholera toxin β subunit-Alexa Fluor 488 fluorescence across all major retinal targets. The density and ration of ipsilateral:contralateral retinal innervation was similar across all retinorecipient target sites and consistent with previous reports .
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[Show abstract][Hide abstract] ABSTRACT: Cone-independent sustained activation of lateral geniculate (LGN) neurons by blue light pulses. (A,B) Multichannel, multiunit recordings from the LGN of a representative red cone knockin mouse (Opn1mwR) showing widespread and sustained neuronal activation in response to 460 nm light pulses (60 s; 8.3×1014 photons/cm2/s). (C) 655 nm light pulses (60 s; 2.6×1015 photons/cm2/s) isoluminant to the 460 nm stimuli for cones evoked much more transient changes in neuronal activity. Traces in (B) and (C) represent the change in multiunit firing (average of four responses) at corresponding recording sites (circles) in panel A; shaded areas represent interstimulus periods of darkness.
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[Show abstract][Hide abstract] ABSTRACT: Single unit spike discrimination. (A) Mean (± SD) waveform of spikes assigned to three single units (corresponding to units 1–3 in Figure 4). (B) Spike patterns of single units from 0.5 s before to 1.5 s after the start of a 460 nm light pulse (8.3×1014 photons/cm2/s). (C) Expanded view of spike traces in (B) at the points marked *. (D) Log interspike interval (ISI) histograms for units 1–3. Histograms show a sharp peak at ISIs between 3 and 5 ms corresponding to spikes fired in bursts and broader peaks at longer ISIs corresponding to epochs of tonic firing.
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[Show abstract][Hide abstract] ABSTRACT: Genetic and immunohistochemical co-labeling of melanopsin retinal ganglion cells. Representative retinal sections from Opn4Cre/+;Z/EG mice co-labeled with a purified rabbit polyclonal antibody raised against an N-terminus epitope of mouse melanopsin . (A) Anti-Opn4-immunofluoresence (red), (B) GFP expression (green), (C) merge. We found 110–130 GFP positive cells/mm2, which amounts to ∼1,500 cells in the adult mouse retina (based on an area of 14 mm2). Of these cells 86.4% were double labeled (white arrows in A) and 10.2% were GFP positive (green arrows) but lacked detectable melanopsin immunostaining, presumably due to a very low level of melanopsin expression undetectable by the antibody. These GFP positive soma were all restricted to the ganglion cell layer and proximal zone of the inner nuclear layer. There were also some sparse cells (2–6/mm2; red arrows) staining only with anti-melanopsin antibody. In these cells GFP is not expressed to a detectable level owing to insufficient Cre function or GFP expression. The dendrites of all GFP or melanopsin immunospositive cells stratified almost equally in both proximal and distal zones of the inner plexiform layer (IPL). Since the M1 type of melanopsin cells primarily stratify in the distal zone of the IPL ,–, the Cre expressing retinal ganglion cells in mice mark both M1 and additional cell types expressing melanopsin.
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[Show abstract][Hide abstract] ABSTRACT: Anatomy of monocular projections of melanopsin ganglion cells. Representative sections (150 µm thick) from unilaterally enucleated Opn4Cre/+;Z/AP mice stained with chromogenic alkaline phosphatase substrate. mRGC innervations to the (A,B) superior colliculus (SC), (C,D) olivery pretectal nuclei (OPN), and (E,F) lateral geniculate nucleus (LGN) are predominantly contralateral. As shown previously the (G) SCN receives bilateral innervation of mRGC from each retina.
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[Show abstract][Hide abstract] ABSTRACT: The melanopsin knockout LGN lacks high amplitude sustained responses. (A,B) Multichannel, multiunit recordings from the lateral geniculate (LGN) of a representative melanopsin knockout mouse (Opn4−/−) showing predominantly transient on and off activations in response to 60 s light pulses (8.3×1014 photons/cm2/s at 460 nm). Traces in (B) represent the change in multiunit firing (average of three responses) at corresponding recording sites (circles) in panel A; shaded areas represent darkness. (C) Anatomical distribution of light responsive cells detected in all Opn4−/− mice investigated, relative to the total number of cells found in each 200 µm×200 µm grid square (based on 520 units recorded in 10 mice).
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[Show abstract][Hide abstract] ABSTRACT: Irradiance coding is deficient in the lateral geniculate (LGN) of Opn4−/− mice. (A) Average response of all light activated LGN neurons recorded in Opn4−/− (blue; n = 217) and Opn1mwR mice (orange; including both “sustained” and “transient” populations; n = 248) to 2 s blue light pulses. (B,C) Quantification of the firing rate of all light responsive LGN cells in Opn4−/− (B) and Opn1mwR (C) mice during the first 500 ms (left) or remainder (500–2000 ms; right) of the light pulse. Symbols indicate mean (± SEM), and lines indicate mean (±95% CI) of the function that best described the response. Note that even though irradiance coding is a unique property of “sustained” neurons (Figure 6), a clear linear relationship between irradiance and firing rate is apparent in the pooled responses of “sustained” and “transient” cells in Opn1mwR mice. Thus, the deficiency of this activity in Opn4−/− mice (B and Figure 6) does not merely reflect our inability to separate “sustained” and “transient” cell types in this genotype.
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[Show abstract][Hide abstract] ABSTRACT: Rod/cone photoreceptors of the outer retina and the melanopsin-expressing retinal ganglion cells (mRGCs) of the inner retina mediate non-image forming visual responses including entrainment of the circadian clock to the ambient light, the pupillary light reflex (PLR), and light modulation of activity. Targeted deletion of the melanopsin gene attenuates these adaptive responses with no apparent change in the development and morphology of the mRGCs. Comprehensive identification of mRGCs and knowledge of their specific roles in image-forming and non-image forming photoresponses are currently lacking. We used a Cre-dependent GFP expression strategy in mice to genetically label the mRGCs. This revealed that only a subset of mRGCs express enough immunocytochemically detectable levels of melanopsin. We also used a Cre-inducible diphtheria toxin receptor (iDTR) expression approach to express the DTR in mRGCs. mRGCs develop normally, but can be acutely ablated upon diphtheria toxin administration. The mRGC-ablated mice exhibited normal outer retinal function. However, they completely lacked non-image forming visual responses such as circadian photoentrainment, light modulation of activity, and PLR. These results point to the mRGCs as the site of functional integration of the rod/cone and melanopsin phototransduction pathways and as the primary anatomical site for the divergence of image-forming and non-image forming photoresponses in mammals.