Measuring HIppocampal Volume in Mouse Brain - Dorsal, Intermediate, Ventral Distinctions?

Lindsey Kitchenham We recently published a study titled "Characterization of Ultrasonic Vocalization-Modulated Neurons in Rat Motor Cortex Based on Their Activity Modulation and Axonal Projection to the Periaqueductal Gray," which involved regional measurements of neuronal density using fluorescently labeled brain sections. The methods we used in this study can be adapted for hippocampal volume measurements in mice, specifically to distinguish between dorsal, intermediate, and ventral hippocampal regions.

Step 1: Tissue Preparation

Prepare coronal brain slices (50 µm thick or less) using a vibratome or microtome. Use a staining technique (e.g., Nissl staining) to clearly visualize hippocampal boundaries.

Align the sections with a standardized mouse brain atlas (e.g., Paxinos and Watson, 2007 or another version). Use anatomical landmarks from the atlas to divide the hippocampus into: Dorsal hippocampus: Intermediate hippocampus: Ventral hippocampus:

Step 2: ROI Annotation

Open Nissl-stained section images in software like ImageJ/Fiji. Use the ROI Manager tool to manually draw boundaries around the hippocampus for each slice. Subdivide the hippocampus into dorsal, intermediate, and ventral parts based on atlas landmarks.

Measure the area of each hippocampal subdivision (dorsal, intermediate, ventral) in pixels for all slices. Convert the pixel areas into micrometers squared (µm²) using the image calibration scale.

Step 3: Volume Calculation

Slice Interval: Record the slice interval (e.g., 50 µm or 0.05 mm for consecutive sections). If every Nth section is analyzed, adjust the slice interval accordingly (e.g., 50 µm × 4 = 200 µm).

Volume Formula: Calculate the volume for each hippocampal subdivision using the formula: Volume (µm³) = Sum of ROIs’ Areas (µm²) × Slice Interval (µm) If every Nth section is analyzed (e.g., every 4th slice), adjust the slice interval accordingly (e.g., 50 µm × 4 = 200 µm).

Separate Volumes: Sum the areas within the respective AP ranges to calculate separate volumes for the dorsal, intermediate, and ventral hippocampus.

Step 4: Statistical Analysis

Group Comparisons: Compare hippocampal volumes between experimental groups (e.g., control vs. stress) for each subdivision.

Relative Contribution: Calculate the proportion of the total hippocampal volume contributed by each subdivision to assess regional differences in sensitivity to stress or affective states.

Let me know if you’d like additional details or further assistance in adapting this method!

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