Fig 1: The D1-like and D2-like subfamilies have opposite effects on GnRH neuronal activity via different signaling pathways. A, B, Calculated δ values for all GnRH cells in that experimental group in beeswarm plot (mean ± SD). Control experiments (right), perturbation experiments (left). Control from Figure 3A in A showing D4R activation [dopamine (1 μm) + D1/5R antagonist (SCH-23390 50 nm) + D2/3R antagonist (sulpiride 20 nm)] and control from Figure 4A in B showing D1-like receptor activation [D1/5R agonist (A 68930 1 nm)]. A, D4R activation failed to inhibit GnRH neurons incubated in PTX (>4 h, 250 ng/ml, wrench symbolizes uncoupling Gαi/o), demonstrating Gαi/o-mediated D4R inhibition (n = 84, N = 3). B, D1/5R activation failed to excite GnRH neurons incubated in CTX (>4 h, 10 ng/ml, wrench symbolizes uncoupling Gαs), demonstrating Gαs-mediated D1/5R inhibition (n = 70, N = 3). Beeswarm plot indicates the spread of individual cell changes in the frequency of calcium oscillations in response to sulpiride+SCH-23390/DA or A 68930 compared with the spread of individual cell changes when pretreated with PTX or CTX, respectively. Nonparametric Mann–Whitney rank test was used to compare two samples (A). Nonparametric Kruskall–Wallis, followed by Dunn’s multiple comparisons test, was used with A 68930 as the reference (B).
Fig 2: Dopamine-mediated inhibition of GnRH neurons is dependent on dopamine D4 receptor. A–D, Representative calcium imaging recordings (left, OD units) and calculated δ values for all GnRH cells in that experimental group in beeswarm plot (mean ± SD; right). A, Blocking D2/D3R (sulpiride 20 nm) did not abolish the dopamine-induced inhibition of GnRH neurons (n = 120, N = 4). B, Activation of D4R using a specific agonist (A 412997 50 nm) inhibited GnRH neuronal activity (n = 99, N = 3). C, Co-application of a D4R antagonist (L-745870 100 nm) prevented the effect of the D4R agonist, demonstrating the specificity of A412997 (n = 93, N = 3). D, Application of another D4R antagonist (PD 168568 50 nm) also prevented the inhibitory effect of the D2-like receptor agonist (quinpirole 50 nm) in GnRH neurons, indicating that the inhibitory effect of quinpirole is mostly mediated through D4R (n = 89, N = 3). Beeswarm plot indicates the spread of individual cell changes in the frequency of calcium oscillations in response to dopamine (DA; from Fig. 1D) compared with the spread of individual cell changes when challenged with antagonist/DA or agonist. Nonparametric Kruskall–Wallis, followed by Dunn’s multiple comparisons test, was used with DA as the reference (A, B) or nonparametric Mann–Whitney rank test when only using two samples were compared (C, D).
Fig 3: Gnat1rd17 mice exhibit decreased TH and DRD4 protein expressions in the retina. Representative immunofluorescence staining (green) of (a) TH and (b) DRD4 protein expression and quantitative analysis of mean fluorescence intensities of (c) TH in the INL and (d,e) DRD4 expressions within all retinal layers in WT and Gnat1rd17 mice, control and with EAU on day 14 after immunization, respectively. Retinal detachments that show retinal folds are indicated by white asterisks. Shown is one of two experiments with similar results. The data are presented as the mean ± SEM of 6 retinas from 6 mice per group. Differences between WT and Gnat1rd17 groups: * p 0.05, ** p 0.01, and *** p 0.001 (unpaired t-test). Blue, Hoechst staining of nuclei. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PSL, photoreceptor segment layer. The scale bar shows 50 μm.
Fig 4: Retinal transcriptomic remodeling in Gnat1rd17 mice. (a) Volcano plot showing DEGs in the neuroretina of Gnat1rd17 mice compared to WT mice. The vertical dot lines define upregulated (log2 fold change 1) and downregulated (log2 fold change −1) DEGs. The red and blue dots indicate all genes at adjusted p 0.01 and p 0.05, respectively. The grey dots represent all genes with an adjusted p 0.05. (b) The top 10 most significantly upregulated and downregulated protein-coding DEGs, as determined by adjusted p-value. (c) Representative Western blot images and relative protein expression of Gnat1 assessed by densitometry. Vinculin was used as a protein-loading control. The data represent the mean ± SEM of 5 neuroretinas of five normal WT and five Gnat1rd17 mice at the age of 10 weeks. The data shown are representative of three independent experiments. ND, not detected. Abbreviations: Cwc22—CWC22 spliceosome-associated protein; Rtkn2—rhotekin 2; Armh4—armadillo-like helical domain-containing 4; Cfap54—cilia and flagella-associated protein 54; Pccb—propionyl Coenzyme A carboxylase, beta polypeptide; Fndc1—fibronectin type III domain-containing 1; Tgm3—transglutaminase 3, E polypeptide; Pdzph1—PDZ and pleckstrin homology domains 1; Pla2g4e—phospholipase A2, group IVE; Lama3—laminin, alpha 3; Gnat1—guanine nucleotide-binding protein, alpha transducing 1; Sik2—salt inducible kinase 2; Abhd14a—abhydrolase domain-containing 14A; Gm4792—predicted gene 4792; Irf7—interferon regulatory factor 7; Haus1—HAUS augmin-like complex, subunit 1; Drd4—DA receptor D4; Mns1—meiosis-specific nuclear structural protein 1; Peli2—pellino 2; Ppargc1b—peroxisome proliferative activated receptor, gamma, coactivator 1 beta.
Fig 5: Dopamine D1-like receptor activation increases the activity of GnRH neurons. A–C, Representative calcium imaging recordings (left, OD units) and calculated δ values for all GnRH cells in that experimental group in beeswarm plot (mean ± SD; right). A, Contrary to the inhibitory effect of dopamine, application of a D1-like receptor agonist (A 68930 1 nm) increased the rate of spontaneous calcium oscillations in GnRH neurons (n = 86, N = 3). B, The stimulatory effect of A 68930 was blocked by the D1-like receptor antagonist (SCH-23390 50 nm), demonstrating the specificity of A 68930 (n = 77, N = 3). C, Blocking the D4R (L-745870 100 nm) did not prevent the excitatory effect of SCH-23390 (n = 77, N = 3). Beeswarm plot indicates the spread of individual cell changes in the frequency of calcium oscillations in response to A 68930 compared with the spread of individual cell changes when maintained in AAB or challenged with antagonist/A 68930. Nonparametric Kruskall–Wallis, followed by Dunn’s multiple comparisons test, was used with A 68930 as the reference.
Supplier Page from Abcam for Anti-DRD4 antibody