Oxytocin and dopamine receptor expression: Cellular level implications for pair bonding

Oxytocin (Oxtr) and dopamine (Drd1, Drd2) receptors provide a canonical example for how differences in neuromodulatory receptors drive individual and species-level behavioral variation. These systems exhibit striking and functionally-relevant differences in nucleus accumbens (NAc) expression across monogamous prairie voles (Microtus ochrogaster) and promiscuous meadow voles (Microtus pennsylvanicus). However, their cellular organization remains largely unknown. Using multiplex in situ hybridization, we mapped Oxtr, Drd1, and Drd2 expression in sexually naïve and mate-paired prairie and meadow voles. Prairie voles have more Oxtr+ cells than meadow voles, but Oxtr distribution across dopamine-receptor cell class was similar, indicating a general upregulation rather than cell class bias. Oxtr was enriched in cells that express both dopamine receptors (Drd1+/Drd2+) in prairie voles, suggesting these cells may be particularly sensitive to oxytocin. We found no species or pairing-induced differences in Drd1+ or Drd2+ cell counts, suggesting prior reports of expression differences may reflect upregulation in cells already expressing these receptors. Finally, we used single-nucleus sequencing to provide the first comprehensive map of Oxtr and Drd1-5 across molecularly-defined NAc cell types in the prairie vole. These results provide a critical framework for understanding how nonapeptide and catecholamine systems may recruit distinct NAc cell types to shape social behavior.

Methods
Images were analyzed using Fiji ImageJ software (version 2.14.0/1.54f). Images were split into four channels:

Channel 0 = DAPI
Channel 1 = Drd1
Channel 2 = Drd2
Channel 3 = Oxtr

Regions of interest (ROIs) outlining DAPI-stained nuclei were automatically generated in Fiji ImageJ. Thresholds for DAPI nuclear staining were manually established to eliminate background and accurately overlay nuclei.

DAPI Mask Validation

Accuracy was verified in 10% of images (one per animal) by comparing experimenter-counted vs. automatic nuclei counts.
Counts differed by ≤5%, supporting the reliability of automatic ROI generation.

Signal Detection & Quantification

The DAPI mask overlay was applied to Drd1, Drd2, and Oxtr images (Fig. 1C).
A white top-hat transformation enhanced contrast, improving bright feature detection.
Collected signal data included:

ROI number (nucleus number)
Minimum, mean, and maximum intensity values
% area (for both 16-bit and 8-bit data)

Data Processing & Analysis

Cellular distribution data was analyzed via custom Python code (GitHub repository).
This script identified:

Positively labeled nuclei for each channel
Double and triple-labeled cells (co-expression)

Oxtr Puncta Quantification

Oxtr puncta were quantified using:

Morphological Filters Plugin (GitHub link)
Object Inspector (2D/3D) Plugin