Journal: The Journal of physiology
Article Title: Interactions of cone cannabinoid CB1 and dopamine D4 receptors increase day/night difference in rod-cone gap junction coupling in goldfish retina
doi: 10.1113/JP281308
Figure Lengend Snippet: In the subjective day simultaneous blockade of both CB1Rs and D4Rs following 30-min blockade of D4Rs decreased photoreceptor tracer coupling to a greater extent than did blockade of CB1Rs alone or blockade of D4Rs alone.(A–D, representative examples of the extent of Neurobiotin tracer diffusion through photoreceptor gap junction channels in the subjective day under four experimental conditions (see text). Following Neurobiotin injection into individual cones in dark-adapted intact goldfish retinas (1 cone/retina/animal), fluorescence was evident in many rods and cones in the subjective day in the presence of SR (B, D-SR) or the D4R antagonist spiperone (SPI, 5 μM) (C, D-SPI). In contrast, fluorescence was evident in only a few photoreceptor cells in the subjective day (A, control, D-CTL) and in the subjective day in the presence of both SPI and SR following a 30-min SPI treatment (D, D-SPI + SR). In each panel (A–D), confocal images of whole-mount retina at the level of rod inner segments are shown on the left, and perpendicular views of the three-dimensional reconstruction of the rods and cones from the same retina are shown on the right. Some cones (triangles) and rods (arrowheads) are indicated. Scale bars: 50 μm. E–G, average numbers of stained photoreceptor cells (both rods and cones, E), rods alone (F) and cones alone (G) following iontophoresis of Neurobiotin into individual cones (1 cone/retina/animal) in the subjective day (control (D-CTL, n = 5), subjective day with SPI (D-SPI, n = 4), subjective day with SR (D-SR, n = 4), subjective day with co-application of SPI and SR (D-SPI + SR, n = 4) following 30-min application of SPI alone, and subjective night with SR (N-SR, n = 5) for each experimental condition). Prior D4R blockade for 30 min in the subjective day reversed the effect of SR141716A in the subjective day; i.e. following D4R blockade, SR141716A decreased rod-cone tracer coupling, an effect that was significantly different from the effects of spiperone alone in the day (E, both rods and cones: P < 0.0001; F, rods alone: P = 0.01287; G, cones alone: P = 0.03814) and SR141716A alone in the day (E, both rods and cones: P = 0.009; F, rods alone: P = 0.01298; G, cones alone: P = 0.04471). In addition, the numbers of coupled photoreceptor cells (including both rods and cones) following co-application of SR141716A and spiperone in the subjective day was not significantly different (E, P = 0.07515) from that observed in the subjective night in the presence of SR141716A alone, suggesting that the effect of endogenous CB1R activation on rod-cone tracer coupling at night (i.e. an increase in coupling) depends on the absence of endogenous D4R activation. F and G, statistical analyses (two-way ANOVA followed by LSD post hoc test; see Methods) were performed to compare the numbers of rods (F) and cones (G) in the four experimental conditions. E–G, data are depicted as means ± SD. Some data points overlap. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. [Colour figure can be viewed at wileyonlinelibrary.com]
Article Snippet: The selective CB 1 R antagonist SR141716A, and the general D2R family selective antagonist spiperone, which at a concentration of 5 μ m selectively blocks cone D 4 Rs in intact goldfish retina ( Ribelayga et al . 2008 ), but not D 1 Rs on cone bipolar cell dendrites ( Chaffiol et al . 2017 ), were purchased from Tocris (Bristol, UK).
Techniques: Diffusion-based Assay, Injection, Fluorescence, Control, Staining, Activation Assay
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![In the subjective day simultaneous blockade of both CB1Rs and D4Rs following 30-min blockade of D4Rs decreased photoreceptor tracer coupling to a greater extent than did blockade of CB1Rs alone or blockade of D4Rs alone.(A–D, representative examples of the extent of Neurobiotin tracer diffusion through photoreceptor gap junction channels in the subjective day under four experimental conditions (see text). Following Neurobiotin injection into individual cones in dark-adapted intact goldfish retinas (1 cone/retina/animal), fluorescence was evident in many rods and cones in the subjective day in the presence of SR (B, D-SR) or the D4R antagonist spiperone (SPI, 5 μM) (C, D-SPI). In contrast, fluorescence was evident in only a few photoreceptor cells in the subjective day (A, control, D-CTL) and in the subjective day in the presence of both SPI and SR following a 30-min SPI treatment (D, D-SPI + SR). In each panel (A–D), confocal images of whole-mount retina at the level of rod inner segments are shown on the left, and perpendicular views of the three-dimensional reconstruction of the rods and cones from the same retina are shown on the right. Some cones (triangles) and rods (arrowheads) are indicated. Scale bars: 50 μm. E–G, average numbers of stained photoreceptor cells (both rods and cones, E), rods alone (F) and cones alone (G) following iontophoresis of Neurobiotin into individual cones (1 cone/retina/animal) in the subjective day (control (D-CTL, n = 5), subjective day with SPI (D-SPI, n = 4), subjective day with SR (D-SR, n = 4), subjective day with co-application of SPI and SR (D-SPI + SR, n = 4) following 30-min application of SPI alone, and subjective night with SR (N-SR, n = 5) for each experimental condition). Prior D4R blockade for 30 min in the subjective day reversed the effect of SR141716A in the subjective day; i.e. following D4R blockade, SR141716A decreased rod-cone tracer coupling, an effect that was significantly different from the effects of spiperone alone in the day (E, both rods and cones: P < 0.0001; F, rods alone: P = 0.01287; G, cones alone: P = 0.03814) and SR141716A alone in the day (E, both rods and cones: P = 0.009; F, rods alone: P = 0.01298; G, cones alone: P = 0.04471). In addition, the numbers of coupled photoreceptor cells (including both rods and cones) following co-application of SR141716A and spiperone in the subjective day was not significantly different (E, P = 0.07515) from that observed in the subjective night in the presence of SR141716A alone, suggesting that the effect of endogenous CB1R activation on rod-cone tracer coupling at night (i.e. an increase in coupling) depends on the absence of endogenous D4R activation. F and G, statistical analyses (two-way ANOVA followed by LSD post hoc test; see Methods) were performed to compare the numbers of rods (F) and cones (G) in the four experimental conditions. E–G, data are depicted as means ± SD. Some data points overlap. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. [Colour figure can be viewed at wileyonlinelibrary.com]](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2046/pmc08882046/pmc08882046__nihms-1772700-f0006.jpg)