A noncanonical inhibitory circuit dampens behavioral sensitivity to light

Abstract

Retinal ganglion cells (RGCs) drive diverse, light-evoked behaviors that range from conscious visual perception to subconscious, non-image-forming behaviors. It is thought that RGCs primarily drive these functions through the release of the excitatory neurotransmitter glutamate. We identified a subset of melanopsin-expressing intrinsically photosensitive RGCs (ipRGCs) in mice that release the inhibitory neurotransmitter γ-aminobutyric acid (GABA) at non-image-forming brain targets. GABA release from ipRGCs dampened the sensitivity of both the pupillary light reflex and circadian photoentrainment, thereby shifting the dynamic range of these behaviors to higher light levels. Our results identify an inhibitory RGC population in the retina and provide a circuit-level mechanism that contributes to the relative insensitivity of non-image-forming behaviors at low light levels.

Fig. 1.. ipRGCs are a potential source of inhibitory input to non-image forming visual brain areas.

(A) Intravitreal injections of AAV2/hSyn-FLEX-Chrimson-tdTomato in Gad2-IRES-Cre mice to label GABAergic cells in the retina. Coronal brain sections were made 1–2 months post infection. (B-D) tdTomato+ axons were consistently observed (14/14 animals) in the IGL (B), vLGN (B) SCN (C), and OPN (D). Scale bar 200 μm. (E) Intravitreal injections of AAV2/hSyn-DIO-mCherry in Gad2-IRES-Cre mice to label GABAergic cells in the retina. Retinas were immunostained for melanopsin to label ipRGCs. (F) Melanopsin and mCherry labeling in Dorsal-temporal (top) and ventral-nasal (bottom) quadrants of retinas in (E) . Solid white circles: Gad2+ ipRGCs and dotted green circles: Gad2− negative ipRGCs. (G) Percentage of melanopsin immunoreactive cells that were Gad2+. n = 7−8 retinas/quadrant. Data are mean ± SD.

Fig. 2.. ipRGCs express Gad2.

(A) In situ hybridization for Opn4 (green) and Gad2 (magenta). ONL (outer nuclear later), INL (inner nuclear layer), GCL (ganglion cell layer). (B) Gad2+ (top) and Gad2− (bottom) ipRGCs from panel A. (C) Strategies for labeling ipRGC axons in the SCN. Black text: reporter mice in which the synaptophysin-tdT fusion protein was expressed in the presence of Cre recombinase (Syp-tdT or Ai34) were intravitreally injected with the pgk-Cre virus. Gray text: Opn4^Cre/+ animals were intravitreally injected with a virus driving Cre-dependent expression of Chrimson-tdT. (D) ipRGC terminals in SCN terminal of Syp-tdt mice intravitreally injected with pgk-Cre virus (top) and immunolabeled for Gad65 (magenta) and synapsin to label axon terminals (cyan). Bottom panels show zoomed in images of ipRGC axon terminals that were Gad65 immunoreactive (boxes 1 and 2) and Gad65 negative (box 3). (E) Percentage of ipRGC terminals that were Gad65 immunoreactive (IR).

Fig. 3.. Functional GABA release by ipRGCs.

(A) SCN acute brain slices were prepared from ChR2-YFP (Ai32) mice eye injected with pgk-Cre virus. Full field 470nm light flashes were used to photo-activate ipRGC axons. (B) Neurobiotin-filled SCN neurons (magenta, indicated by arrows) in SCN slices labeled for VIP. ipRGC axons are labeled in green. (C) Zoomed in images of the VIP− (top panels) and VIP+ (bottom) neurons in B. (D) EPSCs (black) and IPSCs (red) elicited in SCN neurons after photoactivating ipRGC axons in the presence of TTX and 4-AP. The blue line indicates delivery of a 1ms light stimulus. TTX (tetrodotoxin), 4-AP (4-aminopyridine). (E) Synaptic latency of EPSCs and IPSCs following photoactivation of ipRGC axons. n.s. (not significant). (F) Proportion of VIP+ (left) and VIP− (right) SCN neurons that receive excitatory and/or inhibitory input from ipRGCs. (G) Example recording from an SCN neuron that receives both excitatory and inhibitory ipRGC input. Bath application of NBQX and D-APV abolished the EPSC, but did not affect the IPSC. Subsequent application of gabazine abolished the IPSC. (H) EPSC (black, left) and IPSC (red, right) amplitude in SCN neurons receiving both excitatory and inhibitory input from ipRGCs before and following application of NBQX/D-APV and then gabazine. n = 6 cells. All data are mean ± SD.

Fig. 4.. GABA release by ipRGCs influences non-image forming behaviors.

(A) Representative PLR images from control (left panels, Opn4^+/+; Gad2^fx/fx) and Gad2 cKO (right panels, Opn4^Cre/+; Gad2^fx/fx) mice in darkness (top), dim light (middle, 10.9 log quanta/cm²/s) and bright light (bottom, 13.9 log quanta/cm²/s). (B) Control and Gad2cKO pupil area in the dark. (C) Irradiance-response relationship of PLR in control and Gad2 cKO mice. (D) Representative double-plotted actograms from control (top) and Gad2 cKO (bottom) mice. Mice were initially exposed to a 12:12 light dark cycle with 100 lux light during the light phase. The light level was subsequently lowered to 1.5 lux and 0.2 lux. The mice were exposed to a 6-hour phase advance each time the light level was lowered. (E-F) Circadian amplitude measured using the peak amplitude of the X² periodogram (E) and percent activity during the light phase (F) in control (black, n = 8) and Gad2 cKO (red, n = 9) mice. All data are mean ± SD. n.s. (not significant). * P < 0.05, ** P < 0.01 (Mann-Whitney U test).