Advanced Retina Dr. Anthony Vugler UCL-Institute of Ophthalmology Overview: • The discovery of intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) • Structure and function of ipRGCs – Anatomy and physiology – How do ipRGCs contribute to visual function? Rods and cones account for all photoreceptive input to the mammalian CNS… ONL OPL INL IPL GCL Abbreviations: outer nuclear layer (ONL), outer plexiform layer (IPL), inner nuclear layer (INL), ganglion cell layer (GCL). Could there be something else apart from rods and cones? Evidence came from studies of retinal degenerate (rd) mice, which have a mutation in the β subunit of rod-specific phosphodiesterase (PDE). This leads to a rapid degeneration of rods followed by a slower loss of cones. Residual cones In rd retina ONL INL INL GCL GCL Normal retina rd retina rd mice retain a pupillary light reflex (PLR) TABLE 1 TIME OF LATENT PERIOD TIME OF CONTRACTION DIAMETER OF PUPIL AVERAGE CONDITION INDIVIDUAL EYE CONTRAC- OF RETINA TJON Sulfide of 1 2 3 4 5 1 2 3 4 5 Atropin eserine - _.~ -- Gray Q 28 Left Normal 1.46.-0.6160.3 0.3 0.3 0.3 0.3 3.0 3.0 3.0 3.0 3.0 2.31 0.231 Black d’ 23 Left Normal 1.54-0.5390.6 0.6 0.6 0.6 0.6 3.0 3.0 3.3 3.3 3.3 2.31 0.099 Black 8 23 Right Normal 1.54-0.5390.6 0.6 0.6 0.6 0.6 3.0 3.3 3.3 3.3 3.3 2.31 0.099 Gray Q 12 Left Normal 1.54-0.5390.6 0.6 0.6 0.6 0.6 3.0 3.0 3.6 4.2 4.2 2.31 0.924 Gray Q 12 Right Normal 1.54-0.6160.6 0.6 0.6 0.6 0.6 4.2 4.2 4.2 5.6 5.6 2.31 0.924 Gray Q 13 Left Normal 1.54-0.6160.6 0.6 0.6 0.6 0.6 3.6 3.6 3.6 3.6 3.6 2.31 0.385 Gray Q 13 Right Normal 1.54-0.6160.6 0.6-0.6 0.6 0.6 3.6 3.0 3.0 3.0 3.0 2.31 0.385 Gray Q 10 Left Normal 1.54-0.6930.6 0.6 0.6 0.6 0.6 3.0 3.0 3.0 3.0 3.0 2.31 0.154 Gray Q 10 Right Normal 1.54-0.6160.6 '0.6 0.6 0.6 0.6 3.0 3.0 3.0 3.0 3.0 2.31 0.154 Chinchilla Q 11 Left Normal 1.54-0.6160.6$ 0.6. 0.6 0.6-0.6 5.4 5.4 5.4 5.4 6.8 2.16 0.616 Chinchilla Q 11 Right Normal 1.54-0.6160.6-t 0.6+0.6+0.6+0.6+ 4.8 5.4 5.4 5.4 5.4 2.16 0.616 --- 1.53-0.602 0.57 3.73 2.28 0.417 - - -- Gray Q 31 Left Rodless 1.54-0.62 2.4 2.4 2.7 2.7 2.4 2.4 2.4 2.1 2.1 1.8 2.31 0.154 Gray Q 31 Right Rodless 1.54-0.62 3.0 3.3 2.7 3.0 3.0 2.4 1.5 1.5 2.1 1.8 2.31 0.154 Black 8 Left Rodless 2.31-1.16 3.3 3.6 6.0 6.6 Ani nal cl loked to death Chinchilla Q Left Rodless 1.39-0.61 1.8 1.8 1.8 2.4 1.8 3.0 3.0 3.0 3.0 3.0 2.70 0.385 Chinchilla ~31 Left .Rodless 1.39-1.16 1.2 1.5 1.8 1.8 1.8 3.0 2.4 2.4 2.4. 2.4 2.70 0.308 Brown 3 7 Left Rodless 1.93-1.16 1.8 3.0 3.0 3.0 1.8 3.0 2.0 3.0 3.0 2.0 2.39 0.154 Brown 3 7 Right Rodless 1.93-1.16 0.6 0.6 2.4 1.8 2.4 2.4 3.0 1.8 3.0 2.4 2.39 0.154 Brown Q 34 Left Rodless 1.54-0.77 1.8 3.0 3.0 3.0 3.0 2.4 1.8 2.4 3.0 1.8 2.39 0.365 Brown Q 34 Right Rodless 1.54-0.77 1.8 3.0 3.0 3.0 4.8 3.0 1.8 1.8 2.4 1.8 2.31 0.365 Brown Q 6 Left Rodless L.54-1.16 1.8 1.2 1.8 0.6 0.6 3.0 1.8 2.4 2.4 3.6 2.39 0.231 Brown Q 6 Right Rodless ..54-1.16 1.8 1.5 1.2 0.6 0.6 2.4 2.4 2.4 3.6 3.6 2.39 0.231 -- ~ . .65-9.40 2.18 2.56 2.43 0.250 All diameters are given in millimeters. All times are given in seconds. Clyde Keeler noted that rodless animals had a slower and weaker PLR than normals. He concluded that the iris may function independently of vision in rodless animals (based on work in eels from the 1840s) and that the deficits in rodless animals pointed to a regulatory system for iris constriction in normal eyes. (Keeler, (1927) American J. Physiology 81: 107-112). Russell Foster • Studied the photoperiodic response in quail – PhD in the Dept. Zoology University of Bristol • Seasonal gonad maturation is mediated by deep brain photoreceptors in the hypothalamus. – Foster et al., (1985) Nature 313(3): 50-52. – Defined the action spectrum of the opsin involved. Magazine Magazine R597 R597 Birds have deep brain photoreceptors in the hypothalamus ABABC C H H mV PVO PVO mV 20 OT OT 20 5 sec 5 sec -58 mV 3V 3V -58 mV PT 3VPT 3V SE SE (PVO) D D -58 mV -58 mV hv hv 20 mV 20 mV 5 sec 5 secIJIJK K OPN5 70 OPN5 700.25 0.25 Control knockdown -64 mV -64 mV Control knockdown 60 60 0.2 hv hv 0.2 50 50 EFEFG G 0.15 * 40 * 40 * 0.15 * 30 300.1 0.1 mRNA level (nCi) 20 20 mRNA level (nCi) 0.05 0.05 10 TSHB 10 TSHB (5) (8)((5)(5)(8)(8) (5) 8) Number of OPN5 +ve cells/area 0 0 Number of OPN5 +ve cells/area 0 0 Biocytin BiocytinOPN5 OPN5Merge Merge OPN5 OPN5OPN5 OPN5 Control ControlControl Control knockdown knockdownknockdown knockdown Current Biology Current Biology Figure 1. Intrinsic photosensitivityFigure 1. Intrinsic of photosensitivityOPN5-positive CSF-contacting of OPN5-positive neurons CSF-contacting in the paraventricular neurons in the organ paraventricular (PVO). organ (PVO). (A) Schematic drawing(A) Schematic of the quail drawing mediobasal of the hypothalamus. quail mediobasal PVO, hypothalamus. paraventricular PVO, organ; paraventricular PT, pars tuberalis organ; of PT, the pars pituitary tuberalis gland; of the OT, pituitary optic tract; gland; OT, optic tract; 3V, third ventricle. (B)3V, OPN5-positivethird ventricle. CSF-contacting(B) OPN5-positive neurons CSF-contacting in the PVO neurons of 1-day-old in the quail. PVO of(C) 1-day-old Schematic quail. drawing (C) Schematic of representative drawing OPN5-pos of representative- OPN5-pos- itive CSF-contactingitive neurons CSF-contacting in panel (B). neurons S, subependymal in panel (B). layer;S, subependymal E, ependymal layer; layer. E, (D)ependymal Light (hv ,layer. indicated (D) Light by a ( hvbar, indicated below) -evoked by a bar strong below) -evoked strong depolarization anddepolarization fast action potentials and fast in action a CSF-contacting potentials in neuron.a CSF-contacting (E–G) The recordedneuron. (E–G) cell filled The recordedwith biocytin cell showedfilled with OPN5-immunoreactivity. biocytin showed OPN5-immunoreactivity. (H) Light response (H)apparent Light response in normal apparent ACSF (black in normal trace) ACSF persisted (black during trace) bath persisted application during of bath an antagonist application cocktail of an antagonist (50 µM APV, cocktail 50 µM (50 CNQX, µM APV, 50 µM CNQX, 50 µM Picrotoxin) that50 µ blocksM Picrotoxin) conventional that blocks neurotransmission conventional neurotransmission (red trace). (I) Effect (red of ICVtrace). injection (I) Effect of OPN5of ICV siRNAinjection on ofOPN5 OPN5 immunoreactivity siRNA on OPN5 in immunoreactivity in the PVO of eye-patched,the PVO pinealectomized of eye-patched, quail. pinealectomized (J) The number quail. of OPN5-positive(J) The number neuronsof OPN5-positive was significantly neurons reduced was significantly by ICV injection reduced of bysiRNA. ICV injection of siRNA. (K) Long-day-induced(K) Long-day-induced expression of TSHB expression mRNA was of TSHBsignificantly mRNA suppressed was significantly by siRNA-mediated suppressed by OPN5 siRNA-mediated knockdown OPN5in eye-patched, knockdown pinea in eye-patched,- pinea- lectomized quail. TSHBlectomized mRNA quail. levels TSHB were mRNAdetermined levels by were in situdetermined hybridization by in (alsositu hybridizationsee Figure S2). (also *P see< 0.05 Figure (Student’s S2). *P t<-test). 0.05 The(Student’s number t-test). in The number in parentheses indicatesparentheses the number indicates of animals the number used. Values of animals are mean used. + ValuesSEM. Control, are mean Scrambled + SEM. Control, control; Scrambled OPN5 knockdown, control; OPN5 OPN5 knockdown, siRNA.