Confocal Analysis of Cholinergic and Dopaminergic Inputs onto Pyramidal Cells in the Prefrontal Cortex of Rodents

Abstract

Cholinergic and dopaminergic projections to the rat medial prefrontal cortex (mPFC) are both involved in cognitive functions including attention. These neuronal systems modulate mPFC neuronal activity mainly through diffuse transmission. In order to better understand the anatomical level of influence of these systems, confocal microscopy with triple-fluorescent immunolabeling was used in three subregions of the mPFC of rats and Drd1a-tdTomato/Drd2-EGFP transgenic mice. The zone of interaction was defined as a reciprocal microproximity between dopaminergic and cholinergic axonal segments as well as pyramidal neurons. The density of varicosities, along these segments was considered as a possible activity-dependant morphological feature. The percentage of cholinergic and dopaminergic fibers in microproximity ranged from 12 to 40% depending on the layer and mPFC subregion. The cholinergic system appeared to have more influence on dopaminergic fibers since a larger proportion of the dopaminergic fibers were within microproximity to cholinergic fibers. The density of both cholinergic and dopaminergic varicosities was significantly elevated within microproximities. The main results indicate that the cholinergic and dopaminergic systems converge on pyramidal cells in mPFC particularly in the layer V. In transgenic mice 93% of the pyramidal cells expressed the transgenic marker for Drd2 expression, but only 22% expressed the maker for Drd1ar expression. Data presented here suggest that the modulation of mPFC by dopaminergic fibers would be mostly inhibitory and localized at the output level whereas the cholinergic modulation would be exerted at the input and output level both through direct interaction with pyramidal cells and dopaminergic fibers.

Keywords: acetylcholine; cingulate cortex; diffuse transmission; dopamine; infralimbic cortex; prelimbic cortex.

Figure 1

Methodology for quantification of micropoximities with the Leica confocal microscope. Images of the regions of interest (Cg1, PrL, IL) were randomly taken using 100× oil lens in sequential scanning mode on the coronal sections of rats (A, Bregma, +2.7 mm) and mice (B, Bregma, +1.8 mm). Microphotographs were taken every 200 μm, starting at 0.1 mm above the ventral border of the IL cortex to the dorsal border of Cg1 in each layer (A,B). Red squares indicate location of scanning in Layer V, and blue squares indicate location of scanning in Layer II/III. In the BAC Drd1a-tdTomato and GENSAT BAC Drd2-EGFP double transgenic mice, two images were taken in each layer in each subregion of the mPFC (B). The close appositions (within 3 μm in 3D) among cholinergic (green), dopaminergic (blue) fibers, and pyramidal (red) cells were examined on the projection image of three consecutive scanning sections (step size of 1 μm) as well as each single scanning section (C–E). Representative examples of ChAT and TH fibers microproximity are shown (squares in D). Arrows showed examples of pyramidal cells within microproximity of ChAT fibers and TH fibers (E). (White scale bar: 25 μm; Yellow scale bar: 5 μm.

Figure 2

Density of the ChAT (green) fibers (A,B,E,F,I,J) and TH (blue) fibers (C,D,G,H,K,L) in Layer II/III and Layer V of cingulate cortex (Cg1, A–D), prelimbic (PrL, E–H) and infralimbic (IL, I–L) subregions of the medial prefrontal cortex in rats. The density of ChAT fibers was greater than those of TH fibers and showed regional differences. Scale bar: 50 μm.

Figure 3

Representative examples of ChAT/TH fibers within microproximity of each other (arrows) (A–D). The measured lengths of microproximity segments are illustrated by drawing of ChAT (green lines) and TH (blue lines) fibers (A’–D’). Scale bar: 5 μm.

Figure 4

Representative examples of ChAT (green) and TH (blue) varicosities on fiber segments within (A,B) or outside (C–F) the microproximity of the other fiber system in rat mPFC. Note that an increased density of varicosities was seen in segments in microproximity. The number of varicosities counted on microproximity segments are illustrated by indentations on drawing of ChAT (green lines) and TH (blue lines) fibers (A’–F’). Scale bar: 10 μm.

Figure 5

Representative overlay projection images (A–D) of triple immunolabeling of ChAT (green), TH (blue) fibers, and pyramidal cells (red) from three consecutive optic sections in rat mPFC. Arrows show examples of microproximities. Most pyramidal cells were within microproximity of both ChAT and TH fibers. Scale bar: 5 μm.

Figure 6

Colocalization of markers of D₁aRr (A) or D₂Rr (D) expression with EeAAC1 pyramidal cells (B,E) in Drd₁a-tdTomato/Drd₂-EeGFP mice (overlay images, C,F). A majority of pyramidal cells expressed Drd₂ marker (stars in A,C) whereas a minority of pyramidal cells (crosses) expressed Drd1a marker(stars in D,F). Scale bar: 50 μm.

Figure 7

Colocalization of markers of D₁aR (A) or D₂R (D) expression with ChAT (B,E) in Drd₁a-tdTomato/Drd₂-EGFP mice (overlay images, C,F). The colocalization of Drd1a or Drd2 markers and cholinergic fibers was very scarce. Drd₁a and Drd₂ markers were detected in cortical cell soma but not ChAT fibers. Scale bar: 50 μm.