A visual circuit uses complementary mechanisms to support transient and sustained pupil constriction
- Johns Hopkins University, United States
- Harvard Medical School, United States
- Massachusetts General Hospital, United States
- University of Marlyand, United States
- National Eye Institute, National Institutes of Health, United States
- Beth Israel Deaconess Medical Center, Harvard Medical School, United States
Figures
Figure 1 with 2 supplements
The pupillary light response contains two phases: transient and sustained.
(A) Approximate light intensity ranges (lux) at different times of day. (B) Transient constriction in response to a 10 lux overhead stimulus (mean ± SD). Boxes contain representative pupil images at …
Figure 1—figure supplement 1
Experimental setup and light stimulus details.
(A) Environmental light intensity measured in lux across one day (April 2, 2015) in Baltimore, Maryland, USA. The light meter used is unable to measure light intensities below 1 lux, indicated with …
Figure 1—figure supplement 2
Negative-feedback model of PLR decay.
(A) Diagram displaying how the negative feedback model works (7 s light in example) (See Online Methods for step-by-step explanation). The model assumes that packets of light information are …
Figure 2 with 5 supplements
Transient input to ipRGCs is mediated by rods.
(A) Diagram of ipRGC behavioral circuit. (B) Intensity-response curves of the PLR in each of the photoreceptor mutant mouse lines (mean ± SD): wildtype (n = 6), Rod KO (Gnat1-/- n = 6), Melanopsin …
Figure 2—figure supplement 1
Dark-adapted pupil sizes of photoreceptor mutant mouse lines used.
Dark-adapted pupil sizes of all mouse lines used for photoreceptor investigation. Pupil size was recorded before light onset and pupil area (mm2) is reported. No statistical difference was found for …
Figure 2—figure supplement 2
Rods are required for the transient phase of the PLR.
(A) Diagram of the retina labeling the photoreceptors. For experiments in B–D, WT n = 14, Opn4-/- n = 8, Cnga3-/- n = 4, Gnat2-/- n = 7, Cone-DTA n = 7, Gnat1-/- n = 6, Rod-DTA n = 9. (B) Kinetics …
Figure 2—figure supplement 3
Melanopsin is not required for transient PLR in response to environmentally relevant overhead light.
Transient PLR determined under 3 different experimental light conditions. (Left) Blue (474-nm) LED light presented to contralateral eye (1.9 × 1016 photons/cm2/s). (Middle) White halogen light …
Figure 2—figure supplement 4
Rod input to the transient PLR is influenced by cones.
(A) Cartoon representation of a cone and a diagram of its phototransduction cascade. Different aspects of this cascade are disrupted in the various ‘rod-only’ lines we use. (B) Multiple mouse lines …
Figure 2—figure supplement 5
Melanopsin can drive rapid constriction at high light intensities.
Multiple mouse lines with ipRGCs as the only functional photoreceptors (melanopsin-only) or a mouse line with cones as the only functional photoreceptors (cone-only) were tested. For the experiments …
Figure 3 with 3 supplements
Glutamaterigic output provides precise and rapid transient signaling.
(A) Diagram of ipRGC behavioral circuit. (B) Intensity-response curves of the PLR in each of the neurotransmitter mutant mouse lines (Wildtype n = 6) (ipRGC glu. KO: Opn4Cre/+ ; Slc17a6fl/fl n = 4) (…
Figure 3—figure supplement 1
Dark-adapted pupil sizes of neurotransmitter mutant lines used.
Dark-adapted pupil sizes of all mouse lines used for neurotransmitter investigation. Pupil size was recorded before light onset and pupil area (mm2) is reported. ipRGC glutamate KO mice are the only …
Figure 3—figure supplement 2
Description of conditional PACAP allele.
Schematic of the conditional PACAP allele (Adcyap1lox). Boxes indicate exons (1–5). Grey indicates UTR while black indicates protein coding sequence. A single FRT site remains after removal of …
Figure 3—figure supplement 3
PACAP can drive significant constriction within 30s of high light onset.
Transient constriction was monitored in neurotransmitter mutant mice under high light (5000 lux). Data from each mouse is shown with the mean (black bar). ipRGC glutamate KO mice (Opn4Cre/+ ; …
Figure 4 with 4 supplements
Melanopsin/rod synergy supports PLR under persistent conditions.
(A) Diagram of ipRGC behavioral circuit. (B) Intensity-response curves for wildtype and melanopsin knockout mice (Opn4-/-): transient (dotted lines for reference) and sustained (60 min: solid lines) …
Figure 4—figure supplement 1
Melanopsin is required for sustained constriction across the day.
(A) Sustained constriction at 10,000 lux (WT n = 6, only 1 is plotted due to inability to see extremely small pupils in very bright light, Mel. KO n = 6). (B) Time course of pupil constriction under …
Figure 4—figure supplement 2
Viral infection and expression is specific to ipRGCs.
(A) Schematic of the method to activate exclusively ipRGCs using an exogenous GPCR (hM3D(Gq)) and its ligand (CNO). (B) Confocal microscope image showing infection of ipRGCs observed by mCherry …
Figure 4—figure supplement 3
Rods, but not cones, contribute to sustained PLR sensitivity.
(A) PLR intensity-response curves of wildtype and mice with only melanopsin phototransduction intact (‘melanopsin-only’: Gnat1-/-; Gnat2-/- n = 4, Gnat1-/-; Cnga3-/- n = 4, Rod-DTA; Cone-DTA n = 8) …
Figure 4—figure supplement 4
Rods drive the residual sustained pupil constriction observed in the absence of melanopsin.
(A) Sustained PLR dose-responses for wildtype (n = 11), melanopsin knockout (Opn4-/-, n = 12) and mice with only rod phototransduction intact (‘rod-only’: Cnga3-/-; Opn4-/- n = 4) (mean ± SD). …
Figure 5 with 1 supplement
PACAP is essential for the sustained PLR.
(A) Diagram of ipRGC behavioral circuit. (B) PLR intensity-response curves of sustained constriction in mice lacking glutamatergic or PACAPergic neurotransmission (WT n = 6, ipRGC glu. KO n = 4, …
Figure 5—figure supplement 1
PACAP KO mice display similar PLR phenotypes to ipRGC-specific PACAP KO mice.
(A) Intensity-response curves of the transient PLR (30s light) in each of the neurotransmitter mutant mouse lines (Wildtype n = 6)(ipRGC glu. KO: Opn4Cre/+ ; Slc17a6fl/fl n = 4)(PACAP KO: Adcyap1-/- …
Figure 6 with 1 supplement
Model of ipRGC circuit transitions.
(A and B) Heat maps of (A) transient and (B) sustained PLR as duration and intensity vary. Night, dawn/dusk, and daytime light intensities indicated by ticks on right side of plot. (top) Heat maps …
Figure 6—figure supplement 1
Necessity/Sufficiency heat maps for photoreceptor input to pupil constriction.
Heat maps of necessity and sufficiency of each input (top: rods, cones, melanopsin) as stimulus duration and intensity vary. The necessity/sufficiency heat maps for a particular component were …
Videos
Video 1
Pulsatile pupil constriction in the absence of glutamatergic neurotransmission.
This video is at 5x speed. 1000 lux white light (6500K) turns on at approximately 1s.
Tables
Table 1
Description of photoreceptor mutant mouse lines used.
| Mouse line | Genotype | Effect on retinal function | Citations |
|---|---|---|---|
| Rod KO | Gnat1-/- | No rod phototransduction | (Calvert et al., 2000) |
| Rod-DTA | rdta | No rod cell bodies; cones present early in life | |
| Cone KO1 | Cnga3-/- | No cone phototransduction | (Biel et al., 1999) |
| Cone KO2 | Gnat2cpfl3/cpfl3 | No cone phototransduction | (Chang et al., 2006) |
| Cone-DTA | h.red DT-A | Ablation of all M cones; >95% loss of S cones | (Soucy et al., 1998) |
| Melanopsin KO | Opn4-/- | No melanopsin phototransduction | (Lucas et al., 2003) |
| Cone-only | Gnat1-/-; Opn4-/- | No rod/melanopsin phototransduction | |
| Rod-only 1 | Cnga3-/-; Opn4-/- | No cone/melanopsin phototransduction | |
| Rod-only 2 | Gnat2-/-; Opn4-/- | No cone/melanopsin phototransduction | |
| Rod-only 3 | h.red DT-A; Opn4-/- | No cone cells nor melanopsin phototransduction | |
| Melanopsin-only 1 | Gnat1-/-; Cnga3-/- | No rod/cone phototransduction | |
| Melanopsin-only 2 | Gnat1-/-; Gnat2-/- | No rod/cone phototransduction | |
| Melanopsin-only 3 | rdta; h.red DT-A | No rod or cone cell bodies | |
| Red cone KI | Opn1mwred | Cones have shifted sensitivity to red | (Smallwood et al., 2003) |
| Red cone KI; Rod KO | Opn1mwred;Gnat1-/- | Cones have shifted sensitivity to red, no rod phototransduction |
Table 2
Description of neurotransmitter mutant mouse lines used.
| Mouse line | Genotype | Effect on retinal function | Citations |
|---|---|---|---|
| Melanopsin-Cre | Opn4Cre/+ | Cre expression in ipRGCs | (Ecker et al., 2010) |
| Slc17a6-flox | Slc17a6fl/fl | Exon 2 flanked by loxP sites | (Hnasko et al., 2010) |
| ipRGC glutamate KO | Opn4Cre/+; Slc17a6fl/fl | Silences ipRGC glutamatergic release | |
| PACAP KO | Adcyap1-/- | Whole animal PACAP removal | (Hamelink et al., 2002) |
| PACAP-flox | Adcyap1fl/fl | Exon 2 flanked by loxP sites | See Figure 3—figure supplement 2 |
| ipRGC PACAP KO | Opn4Cre/+ ; Adcyap1fl/- | Silences ipRGC PACAP release |
