Fig. 1 Cx36 at rod/cone contacts. (A) Four-channel labeling of wild-type mouse (B6) retinal section. Cones labeled for cone arrestin (cARR green), rod spherules for the vesicular glutamate transporter 1 (vGluT1, blue), and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) (gray). For clarity, only Cx36 (red) in right half. Cx36 labeling is very dense in the IPL, less so in the OPL. Note that cone pedicles, rod spherules, and Cx36 contained in the OPL. Scale bar, 50 μm. (B) In whole-mount retina, OPL shows Cx36 plaques (red) associated with cone telodendria (green). Scale bar, 10 μm. (C) High magnification (Zeiss LSM800 Airyscan), wild-type mouse retina showing Cx36 plaques (red) associated with cone telodendria (green, arrows) and distinctly beneath the cone pedicles (circled). Colocalization (white) highlights Cx36 on telodendria, not underneath cone pedicles (circled). Colocalized Cx36/cone pedicle sites are contained within the band of rod spherules (vGlut1, blue). Scale bar, 10 μm, applies to all. (D) Left; Representative example shows three rod spherules (blue) and cone telodendria (green) with multiple Cx36 puncta (1 to 4, red) at each contact. Right: No blue channel for clarity. Scale bar, 1 μm, applies to both.
Fig. 2 Cx36 distribution in pan- and conditional-Cx36 knockouts. (A) OPL of wild type (WT; B6), and Cx36 mutants labeled for Cx36 (red) and cone arrestin (green). Top row: cell nuclei stained with DAPI (cyan). Bottom row: Cx36 only for clarity. Note the absence of Cx36 in the pan-Cx36 KO and large reduction in cone- or rod-Cx36 XO. Cx36 beneath cone pedicles (white circles) associated with bipolar cell dendrites, not cones. Scale bars, 10 μm, applies to all. (B) Quantification of Cx36 in the OPL for wild type and Cx36 mutants; individual values (black circles), means (bars), SEM (error bars), and n = number of animals. Statistical test between mutants and respective control littermates (ctl), nonparametric Kruskal-Wallis analysis of variance (ANOVA). (C) Cx36 plaques in the OPL per cone pedicle for wild type and Cx36 mutants. Analysis performed on 7 to 15 sections (45 μm by 45 μm by 0.4 μm) spanning the OPL. Cx36 underneath cone pedicles excluded from the analysis. Wild-type column included B6 mice and mutant littermates (mix). Presentation as in (B). Only statistically significant differences shown (P < 0.05), nonparametric Kruskal-Wallis ANOVA. (D) Averaged median volume of the Cx36 puncta identified in (C), presentation as in (B).
Fig. 3 Cx36 requirement and routes of electrical coupling between photoreceptors. (A to C) Schematic representation of the experimental setup and visualization of the photoreceptors simultaneously patch-clamped and filled with Lucifer yellow through the two recoding pipettes. Simultaneous patch clamp recording of photoreceptor pairs in the living mouse retinal slice are illustrated for a pair of rods (A), a rod/cone pair (B), and a cone/cone pair (C). (D to F) Examples of simultaneous voltage clamp recordings from rod/rod (D), cone/cone (E), and rod/cone (F) pairs obtained in pan-Cx36 KO retinas (red traces) and their respective wild-type control littermates (black traces). Transjunctional current traces in response to 50-ms voltage steps, 10-mV increments from −50 to +50 mV, and the voltage-current relationship whose slope gives an estimate of the transjunctional conductance are shown. (G to I) Rod/rod (G), rod/cone (H), and cone/cone (I) coupling conductances in wild-type (B6) and Cx36 mutant mice. Box plots show the median value (center line), the lower (25%) and upper (75%) quartiles, and minimum and maximum (whiskers). Statistical test: nonparametric Kruskal-Wallis ANOVA. ns, not significant.
Fig. 4 The rod/cone gap junction is modulated by dopamine. (A) Effects of the D2-like dopamine receptor antagonist spiperone on direct rod/rod coupling. Electrical coupling between pairs of adjacent rods was recorded in the cone-Cx36 XO retina. No increase in coupling was observed when spiperone (10 μM) was present in the superfusion and applied for >10 min, indicating that spiperone has no effect on direct rod/rod coupling. (B) Effects of the D2-like dopamine receptor antagonist spiperone and of the agonist quinpirole on rod/cone coupling. Electrical coupling between rod/cone pairs was recorded in wild-type (B6) retinas. Spiperone (10 μM; applied for >10 min, top) significantly increased rod/cone coupling, whereas quinpirole (1 μM; applied for >10 min, bottom) significantly decreased rod/cone coupling. Note the break of the x axis on the spiperone figure: The outlier value is 6040 pS. Numbers represent median value of the conductance [interquartile]. KW, Kruskal-Wallis.
Fig. 5 The rod/cone gap junction is the entry of a major functional pathway. (A) Patch-clamp recordings of the light responses of cones obtained in wild-type and mutant retinas. Wild-type cone responses to brief flashes of light show two components: A fast and transient component (black arrow) and a slowly developing and recovering one (white arrow). Only the fast and transient component is present in mutant retinas, whereas the slow component is eliminated. We conclude that the fast component represents the cone intrinsic response and that the slow component represents the rod-mediated or cone extrinsic response via rod/cone gap junctions. (B) Average intensity-response curves of cones recorded under the conditions depicted in (A). Response is peak amplitude (in mV). Means ± SEM are shown. n = 4 to 8 cells per genotype. Threshold (T, vertical lines) is intensity to elicit a 1-mV response. (C) Average intensity-response curves of cones recorded under the conditions depicted in (A). Response is the area of the hyperpolarization [in arbitrary units (AU)]. Means ± SEM are shown. n = 5 to 8 cells per genotype. Threshold (T, vertical lines) shows intensity to elicit 200 AU.
Fig. 6 Biophysical model of the photoreceptor network in mouse retina. (A) Model is based on a 30:1 rod/cone ratio. Simulated photocurrent functions are illustrated. (B) Cone voltage responses as a function of the rod/cone junctional conductance. The computational model recapitulates the wild-type cone light responses. (C) Intensity-response curves of cones computed for values of rod/cone junctional conductance ranging from 0 to 1000 pS. Note the decrease in threshold and increase in amplitude when the conductance increases. (D) Rod voltage responses as a function of the rod/cone junctional conductance and (E) intensity-response curves of rods computed for values of rod/cone junctional conductance ranging from 0 to 1000 pS. Note that rod/cone coupling has little effect on the response properties. (C and E) Dotted line shows criterion for threshold (1 mV).
- Table 1 Summary of the junctional conductance measurements.
Summary of the junctional conductance measurements.. Data shown are the number of pairs recorded n (number of uncoupled pairs/number of coupled pairs) and the median value [interquartile] of rod/rod, rod/cone, and cone/cone transjunctional conductances measured in wild type [WT (B6)], pan or conditional knockouts, and their respective control littermates. A pair was considered coupled when conductance is >50 pS. Statistical differences between the mutant lines and their respective control groups are indicated (*P < 0.05), nonparametric Kruskal-Wallis ANOVA. The median values of the four WT/control groups were averaged, and the mean (SEM) is shown in the bottom row. See figs. S9 and S10 for more details.
n Rod/rod coupling
(pS)n Rod/cone coupling
(pS)n Cone/cone
coupling (pS)WT (B6) 21(2/19) 135 [85,219] 15(0/15) 453 [219,560] 11(1/10) 72 [26,234] Rod-Cx36 XO 7(6/1) 0 [0,0]* 7(5/2) 0 [0,1]* 6(2/4) 56 [9,114] Rod-Cx36 ctl 7(0/7) 139 [102,254] 11(1/10) 213 [128,574] 7(3/4) 65 [0,126] Cone-Cx36 XO 21(15/6) 0 [0,3]* 7(5/2) 0 [0,4]* 5(2/3) 3 [0,5]* Cone-Cx36 ctl 7(2/5) 108 [52,111] 12(0/12) 313 [156,654] 20(4/16) 46 [5,224] Pan-Cx36 KO 8(6/2) 0 [0,21]* 6(6/0) 0 [0,0]* 13(6/7) 10 [0,38]* Pan-Cx36 ctl 14(2/12) 129 [46,161] 7(1/6) 249 [104,306] 20(3/17) 53 [32,129] Mean WT/ctl 4 128 (7) 4 307 (53) 4 59 (6) - Table 2 List of the primary antibodies used in this study.
Antibody Immunogen Source Concentration Connexin35/36
mouse
monoclonal
antibody,
clone 8F6.2Recombinant
perch
connexin35Chemicon,
catalog no.
MAB30451:1000 Cone arrestin
(cArr) rabbit
polyclonal
antibodySynthetic
peptide,
C-terminal
region of rat
and mouse cArrMillipore,
catalog no.
AB152821:500 Choline acetyl
transferase
(ChAT) goat
polyclonal
antibodyHuman
placenta
enzymeChemicon,
catalog no.
AB144P1:100 Vesicular
glutamate
transporter
type I
(VGLUT1)
Guinea pig
polyclonal
antibodyRecombinant
rat VGLUT1
(amino acids
456 to 500)Synaptic
Systems,
catalog no.
1353041:3000 Enhanced
fluorescent
green protein
(eGFP)
chicken
polyclonal
antibodyRecombinant
GFP emulsified
in Freund’s
adjuvantAves Labs Inc.,
catalog no.
GFP-10101:100 S-cone opsin
(blue cone
opsin) rabbit
polyclonal
antibodyRecombinant
human S-opsinMillipore,
catalog no.
AB54071:400 Red fluorescence
protein (RFP)
rabbit
polyclonal
antibodyRFP fusion
protein
(full-length
amino acid
sequence)
derived from
the mushroom
polyp coral
DiscosomaRockland
antibodies
and assays,
catalog no.
600-401-3791:500
Supplementary Materials
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/28/eaba7232/DC1
Additional Files
Supplementary Materials
Molecular and functional architecture of the mouse photoreceptor network
Nange Jin, Zhijing Zhang, Joyce Keung, Sean B. Youn, Munenori Ishibashi, Lian-Ming Tian, David W. Marshak, Eduardo Solessio, Yumiko Umino, Iris Fahrenfort, Takae Kiyama, Chai-An Mao, Yanan You, Haichao Wei, Jiaqian Wu, Friso Postma, David L. Paul, Stephen C. Massey*, Christophe P. Ribelayga
This PDF file includes:
- Figs. S1 to S13
- Table S1
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