Neurochemical Characterization of Dopaminoceptive Cells in Song Control Nuclei of Canaries and Their Activation During Song Production: A Multiplex Fluorescent In Situ Hybridization Study

Abstract

Highly sensitive in situ hybridization procedures (RNAScope) were used to quantify the expression of three dopamine receptors (Drd1, Drd2, and Drd3) in two song control nuclei (HVC and the Area X of the basal ganglia) that are known to receive dopaminergic inputs and in the periaqueductal gray (PAG) of male and female canaries. Both sexes were treated with testosterone to ensure they would sing actively. We also determined the excitatory versus inhibitory phenotype of the cells expressing these receptors as well as their activation following a period of song production. The three receptor types were identified in each brain area, with the exception of Drd3 in Area X. The density of cells expressing each receptor varied as a function of receptor type and brain area. Surprisingly few sex differences were detected; they do not seem to explain the sex differences in testosterone-induced song. Overall, the density of Drd-positive cells was much lower in PAG than in the two song control nuclei. In HVC, the majority of cells expressing the three receptor subtypes were VGlut2-positive, whereas colocalization with Vglut2 occurred in few cells in Area X and in an intermediate proportion of cells in PAG. The number of inhibitory cells expressing dopamine receptors was limited. Most dopaminoceptive cells in Area X did not express either excitatory or inhibitory markers. Finally, cellular activation during singing behavior, as measured by the expression of Egr1, was observed in cells expressing each of the three dopamine receptor subtypes, except Drd3 in the PAG.

Keywords: Area X; HVC; RRID:SCR_002285; RRID:SCR_004870; dopamine receptor; excitatory neurons; sex differences; singing behavior.

Figure 1.. Photomicrographs illustrating the colocalization of the three dopamine receptors with the markers of excitatory (VGlut2) or inhibitory (Gad2) neurotransmission and with the immediate early gene Egr1 in a male HVC.

DAPI was used to mark cell nuclei in all sections. Panels A to C respectively illustrate the colocalizations with Drd1, Drd2 and Drd3, panel D summarizes the color codes used in the photomicrographs. Magnification bar = 50 μM in the 3 large panels and 25 μm in the small panels illustrating the cellular localization of the labels.

Figure 2.. Relative expression of dopamine receptor types in the HVC of males and females.

(A) Percentage of cells within a 200 × 200 μM square in HVC that had fluorescent label for the 3 dopamine receptor subtypes. (B) Density of cells (numbers/mm²) that expressed the mRNA of each the dopamine receptor subtype. All bar plots represent means + SEM of all data points which are also indicated separately. a, b= p<0.05 vs. Drd2 or Drd3 respectively (collectively for both sexes). (C) Example photomicrographs of receptor densities in males and females. DAPI-labeled nuclei are blue and dopamine receptor expression is labeled in green. White scale bars indicate 100 μm.

Figure 3.. The majority of HVC cells that express dopamine receptors are excitatory.

A) Percentage of cells that expressed fluorescent label for one of the dopamine receptor subtypes colocalized with VGlut2, a marker for excitatory neurons, or B) with Gad2, the marker of inhibitory neurons. All bar plots represent means + SEM of all data points that are also indicated separately. a, b= p<0.05 vs. Drd2 or Drd3 respectively (collectively for both sexes).

Figure 4.. Similar proportions of cells expressing dopamine receptor subtypes are active during song in HVC.

Number of cells that had fluorescent label for one of the dopamine receptor subtypes colocalized with the immediate early gene Egr1. All bar plots represent means + SEM of all data points that are also indicated separately.

Figure 5.. D1 and D2 dopaminoceptive cells in Area X and their colocalization with the neurotransmitter markers VGlut2 and Gad2 and with Egr1.

(A) Density of cells that expressed the mRNA of each of the dopamine receptor subtypes.(B-C) Percentage of Drd1+ or Drd2+ cells that expressed fluorescent label for Vglut2, a marker for excitatory neurons (B) or for Gad2, a marker of inhibitory neurons (C). (D) Percentage of Drd1+ and Drd2+ cells that expressed Egr1. All bar plots represent means + SEM of all data points that are also indicated separately. a= p<0.05 vs. Drd2 (collectively for both sexes).

Figure 6.. Colocalization of Drd1 and Drd2 with VGlut 2, GAD2 and Egr1 in Area X

All photomicrographs come from male sections. The 9 panels illustrate the colocalization of Drd1 (A, C, E) and Drd2 (B, D, F) in green with respectively VGlut2 (A, B) in orange, GAD2 (C, D) in red and Egr1 (E,F) in pink. Magnification bar = 100 μm.

Figure 7.. Relative density of dopaminoceptive cells in the medial PAG and their colocalization with the neurotransmitter markers Vglut2 and Gad2 and Egr1.

(A). Schematic drawing of the ventral part of the brain at the level of the PAG illustrating the distribution of tyrosine hydroxylase positive cells (black dots) and localization of the area where quantification of dopamine receptors was performed (red square). (B) Number of cells that expressed the mRNA of each of the dopamine receptors. (B-C-D) Percentage of Drd+ cells that expressed fluorescent label for Vglut2, a marker for excitatory neurons (C) or for Gad2, a marker of inhibitory neurons (D). (E) Percentage of Drd+ cells that expressed Egr1. All bar plots represent means + SEM of all data points that are also indicated separately. a, b= p<0.05 vs. Drd2 or Drd3 respectively (collectively for both sexes). Abbreviations in panel A: A8–A11, dopaminergic cell groups (A11 corresponds to PAG); EW: nucleus of Edinger-Westphal; FLM: fasciculus longitudinalis medialis; ICO: nucleus intercollicularis; MLD: nucleus mesencephalicus medialis, pars dorsalis; OMd-v: occulomotor nucleus dorsal-ventral; PAG: periqueductal gray; V Ventricle.

Figure 8.. Photomicrographs illustrating the densities of Drd1, Drd2, VGlut 2, GAD2 and Egr1 in the medial part of PAG.

All photomicrographs come from female sections. The 12 panels illustrate the colocalization of Drd1 (A, D, G, J), Drd2 (B, E, H, K) and Drd3 (C, F, I, L) in green with respectively VGlut2 (D, E, F) in orange, GAD2 (G, H, I) in red and Egr1 (J, K, L) in pink. Magnification bar = 100 μm.

Figure 9.. Comparison of the density of dopamine receptors and colocalization with VGlut2, Gad2 or Egr1 across the 3 investigated brain regions.

a=p<0.05 versus Area X; b=p<0.05 vs. PAG by Benjamini –Hochberg corrected multiple t tests.