Cellular Profiles of Prodynorphin and Preproenkephalin mRNA-Expressing Neurons in the Anterior Olfactory Tubercle of Mice

Abstract

The olfactory tubercle (OT) is a striatal region that receives olfactory inputs. mRNAs of prodynorphin (Pdyn) and preproenkephalin (Penk), precursors of dynorphins and enkephalins, respectively, are strongly expressed in the striatum. Both produce opioid peptides with various physiological effects such as pain relief and euphoria. Recent studies have revealed that OT has anatomical and cytoarchitectonic domains that play different roles in odor-induced motivated behavior. Neuronal subtypes of the OT can be distinguished by their expression of the dopamine receptors D1 (Drd1) and D2 (Drd2). Here, we addressed whether and which type of opioid peptide precursors the D1- and D2-expressing neurons in the OT express. We used multiple fluorescence in situ hybridization for mRNAs of the opioid precursors and dopamine receptors to characterize mouse OT neurons. Pdyn was mainly expressed by Drd1-expressing cells in the dense cell layer (DCL) of the OT, whereas Penk was expressed primarily by Drd2-expressing cells in the DCL. We also confirmed the presence of a larger population of Pdyn-Penk-Drd1 co-expressing cells in the DCL of the anteromedial OT compared with the anterolateral OT. These observations will help understand whether and how dynorphins and enkephalins in the OT are involved in diverse odor-induced motivated behaviors.

Keywords: DARPP-32; dopamine receptor D1; dopamine receptor D2; medium spiny neurons; olfactory tubercle; opioids; preproenkephalin; prodynorphin.

Figure 1

Single Probe in situ hybridization (ISH) for Pdyn and Penk in the mouse striatum. Pictures show coronal sections of the anterior OT [approximately at Bregma +1.70 mm of Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates (Franklin and Paxinos, 2008)] except for the lower right panel showing the hypothalamus (approximately at Bregma −1.58 mm). Upper left panel; Pdyn, Upper right panel Penk, Lower panels; Pomc. OT, olfactory tubercle; NAc, nucleus accumbens; dStr, dorsal striatum.

Figure 2

Magnified view of the single probe ISH for Drd1, Drd2, Pdyn, and Penk in the anterior OT. Pictures show coronal sections of the anterior OT (approximately at Bregma +1.70 mm). In the OT, ISH signals of Pdyn and Penk were observed in the DCL. Regions delineated by yellow lines are the Cap regions. Regions delineated by green lines are the ICj. Adjacent sections from one mouse were used for the four pictures. Brain sections in this figure were sampled from a different mouse used in Figure 1.

Figure 3

Pdyn-Penk-Drd1 co-expressing cell cluster in the anterior ventral striatum. (A) Single probe ISH for Pdyn, Penk, Drd1, and Drd2. The pictures show coronal sections of the anterior OT and NAc (approximately at Bregma +1.94 mm). Regions delineated by red lines are a cluster of Pdyn-Penk-Drd1 co-expressing cells. Drd2 signals were not observed in the cluster. Adjacent sections from one mouse were used for the four pictures. Brain sections in this figure (A) were sampled from the same mouse used in Figure 2. (B) Triple fluorescence ISH for Pdyn-Penk-Drd1 (Upper panels) and Pdyn-Penk-Drd2 (Lower panels). Regions delineated by white lines are a cluster of Pdyn-Penk-Drd1 co-expressing cells which did not express Drd2.

Figure 4

Triple fluorescence ISH for Pdyn-Penk-Drd1 mRNAs in the anteromedial and anterolateral OT DCL. Fluorescence images of Pdyn (red), Penk (blue), and Drd1 (green) mRNA signals in the same region. White arrowheads show the colocalization of Pdyn-Penk-Drd1 mRNAs. Right panels, anteromedial DCL; left panels, anterolateral DCL.

Figure 5

Triple fluorescence ISH for and Pdyn-Penk-Drd2 mRNAs in the anteromedial and anterolateral OT DCL. Fluorescence images of Pdyn (red), Penk (blue), and Drd2 (green) mRNA signals in the same region. A white arrowhead shows the colocalization of the Pdyn-Penk-Drd2 mRNAs. White-outlined arrowheads indicate the colocalization of Pdyn-Penk signals, which did not colocalize with Drd2 signals. Right panels, anteromedial DCL; left panels, anterolateral DCL.

Figure 6

Quantification of Pdyn, Penk, Drd1, and Drd2-expressing cells and their colocalization in the anteromedial and anterolateral OT DCL. (A) The percentages were calculated by dividing the number of Pdyn. Penk, Drd1, and Drd2-expressing cells by the number of the DAPI(+) cells in the region of interest. The data of Pdyn and Penk were obtained from Pdyn-Penk-Drd1 co-staining images as shown in Figure 4. (B–D), The percentages of double-positive cells (e.g., Pdyn/Penk shows the percentage of Pdyn-Penk double-positive cells among Penk positive cells in the region of interest). The data in B were obtained from Pdyn-Penk-Drd1 co-staining images as shown in Figure 4. (E) The percentage of triple-positive cells (e.g., Pdyn(+)-Penk(-)/Drd1 shows the percentage of the number of Pdyn(+) and Penk(-) Drd1-expressing cells by the number of total Drd1 positive cells in the region of interest). Bars in the graphs represent mean ± SD. p-Values were calculated by post-hoc Tukey's test after two-way ANOVA. n = 3 mice.

Figure 7

DARPP-32 expression by the putative Pdyn-Penk-Drd1 co-expressing cells in the cell cluster between OT and NAc. Fluorescence images of DARPP-32 immunoreactivity (upper left, green), Penk (upper right, blue), Drd1 (middle left, red) mRNA signals, DAPI (lower left, magenta), and color merged view (middle right; DARPP-32-Drd1-Penk, lower right; DARPP-32-DAPI) in the same region. Regions delineated by white lines are a cluster of putative Pdyn-Penk-Drd1 co-expressing cells. OT, olfactory tubercle; NAc, nucleus accumbens; ICj, Islands of Calleja; VON, ventral olfactory nucleus.

Figure 8

DARPP-32 expression by the putative Pdyn-Penk-Drd1 co-expressing cells in the anteromedial DCL. Fluorescence images of DARPP-32 immunoreactivity (upper left, green), Penk (upper right, blue), Drd1 (middle left, red) mRNA signals, DAPI (lower left, magenta), and color merged view (middle right; DARPP-32-Drd1-Penk, lower right; DARPP-32-DAPI) in the same region. White arrowhead shows the colocalization of the DARPP-32 immunoreactivity and Penk-Drd1 mRNAs. White-outlined arrowheads indicate the colocalization of Penk-Drd1 signals, which did not colocalize with DARPP-32 immunoreactivity.