DOI: 10.5061/dryad.s1rn8pkk3
title: Data from: Oxytocin and dopamine receptor expression: Cellular level implications for pair bonding

description: 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.
description: 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
description: # Oxytocin and dopamine receptor expression: Cellular Level implications
 for pair bonding Dataset DOI:
 [10.5061/dryad.s1rn8pkk3](10.5061/dryad.s1rn8pkk3) ## Description of the
 data and file structure **Data Description** This dataset was collected
 from four cohorts of voles: * Sexually naive prairie voles (NP) * Sexually
 naive meadow voles (NM) * Pairbonded prairie voles (PP) * Paired meadow
 voles (PM) Data was obtained through microscopy image analysis using Fiji
 ImageJ software. **Data Collection & Processing** **Microscopy
 Analysis** * DAPI masks were generated using the Analyze Particles
 function in Fiji ImageJ. Each region of interest (ROI) represents a
 nucleus. * The presence or absence of transcript was assessed for each ROI
 across three channels: D1DR (Drd1), D2DR (Drd2), and OXTR (Oxtr). * Data
 collected per ROI included: * ROI number * ROI area * Mean, minimum, and
 maximum gray values * Percent area covered by pixels **Oxtr Puncta
 Quantification** * Oxtr puncta counts were recorded in the Oxtr Puncta
 Counts file for all four cohorts. * Secondary Object Count (Sec_Object
 Count) was collected only for the OXTR channel, representing the number of
 puncta per ROI. * Puncta detection utilized: * Morphological Filters
 Plugin ([GitHub link](https://github.com/ijpb/MorphoLibJ)) * Object
 Inspector (2D/3D) Plugin **Regional Classification** * Each ROI was
 assigned a Region1 category: * Core1 = Core of the nucleus accumbens *
 Shell1 = Medial shell * Shell3 = Lateral shell * The Region variable
 specifies subregion and hemisphere (left/right). * Rostral-caudal levels
 (RC levels) of the nucleus accumbens were recorded. **Files and
 variables** **File: R3_L1_PM_cellular_distribution_8_bit_v4.xlsx**
 **Description:** rostral-caudal level 1, paired meadow voles, transcript
 distribution data (8 bit) ##### Variables * Region1 (nucleus accumbens
 subregion), * Region (nucleus accumbens subregion with laterality
 indicated), * Channel (label for transcript examined in that wavelength) ,
 * ROI (region of interest = nucleus) , * Area (of nucleus/ROI), * Mean
 (gray value of transcript in that ROI in that channel indicated), * Min
 (gray value of transcript in that ROI in that channel indicated), * Max
 (gray value of transcript in that ROI in that channel indicated), * % Area
 (amount of area with nonzero pixels within ROI for that channel), * Sec
 Object Count (Oxtr puncta for OXTR channel only). Values are blank for
 D1DR and D2DR channels in this column. **File:
 R3_L1_NM_cellular_distribution_8_bit_v3.xlsx**
 **Description:** rostral-caudal level 1, naive meadow voles, transcript
 distribution data (8 bit) ##### Variables * Region1 (nucleus accumbens
 subregion), * Region (nucleus accumbens subregion with laterality
 indicated), * Channel (label for transcript examined in that wavelength) ,
 * ROI (region of interest = nucleus) , * Area (of nucleus/ROI), * Mean
 (gray value of transcript in that ROI in that channel indicated), * Min
 (gray value of transcript in that ROI in that channel indicated), * Max
 (gray value of transcript in that ROI in that channel indicated), * % Area
 (amount of area with nonzero pixels within ROI for that channel), * Sec
 Object Count (Oxtr puncta for OXTR channel only). Values are blank for
 D1DR and D2DR channels in this column. **File:
 R3_L1_NP_cellular_distribution_8_bit_v3.xlsx**
 **Description:** rostral-caudal level 1, naive prairie voles, transcript
 distribution data (8 bit) ##### Variables * Region1 (nucleus accumbens
 subregion), * Region (nucleus accumbens subregion with laterality
 indicated), * Channel (label for transcript examined in that wavelength) ,
 * ROI (region of interest = nucleus) , * Area (of nucleus/ROI), * Mean
 (gray value of transcript in that ROI in that channel indicated), * Min
 (gray value of transcript in that ROI in that channel indicated), * Max
 (gray value of transcript in that ROI in that channel indicated), * % Area
 (amount of area with nonzero pixels within ROI for that channel), * Sec
 Object Count (Oxtr puncta for OXTR channel only). Values are blank for
 D1DR and D2DR channels in this column. **File:
 R3_L1_PP_cellular_distribution_8_bit_v3.xlsx**
 **Description:** rostral-caudal level 1, pairbonded prairie voles,
 transcript distribution data (8 bit) ##### Variables * Region1 (nucleus
 accumbens subregion), * Region (nucleus accumbens subregion with
 laterality indicated), * Channel (label for transcript examined in that
 wavelength) , * ROI (region of interest = nucleus) , * Area (of
 nucleus/ROI), * Mean (gray value of transcript in that ROI in that channel
 indicated), * Min (gray value of transcript in that ROI in that channel
 indicated), * Max (gray value of transcript in that ROI in that channel
 indicated), * % Area (amount of area with nonzero pixels within ROI for
 that channel), * Sec Object Count (Oxtr puncta for OXTR channel only).
 Values are blank for D1DR and D2DR channels in this column. **File:
 R3_L1_Oxtr_Puncta_Counts_v2.xlsx** **Description:** Oxtr puncta counts per
 ROI for naive prairie voles, naive meadow voles, pairbonded prairie voles,
 and paired meadow voles ##### Variables * Animal # (sex is M or F and
 number is corresponding number given in colony) * Species (meadow or
 prairie vole) * Bond status (sexually naive or paired(bonded) * RC level
 (rostral caudal of nucleus accumbens; only level 1 is in this data set) *
 Region1 (subregion of nucleus accumbens) * Region (subregion of nucleus
 accumbens with laterality indicated) * ROI (region of interest corresponds
 to each nuclei detected) * Sec Object Count (# of Oxtr puncta detected
 within ROI) * Volume (^3) (data not used) **Code/software** Data files are
 in Excel sheets. Data sorting was done through Python scripts found on
 Donaldson Lab Github