Oxytocin and Dopamine Receptor Expression: Cellular Level Implications for Pair Bonding

Dataset DOI: 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)
  • 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.

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Methods (From Manuscript)

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

Files and variables

File: R3_L1_PM_cellular_distribution_8_bit_v2.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_v2.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_v2.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_v2.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