302 - Sex-specific Hippocampal Interneuron Population Size and Ntrk2 Expression During Development

Publication Number: 4296.302

Yigit C. Erkus, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Feyza Cetin, Washington University in St. Louis School of Medicine, St. Louis, MO, United States; Furkan Camci, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Nur Aycan, University of Wisconsin, Madison, WI, United States; Nursena Yabalar, University of Wisconsin, Madison, WI, United States; Beiya Zhang, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Kulsum Husain, University of Wisconsin-Madison, Madison, WI, United States; Maxwell Bogdanoff, University of Wisconsin - Madison, Sherman Oaks, CA, United States; Madeline E. Krolak, University of Wisconsin Madison, Chicago, IL, United States; Rozerin G. Yuksel, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Sefer Yapici, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Andre Sousa, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Ryan Risgaard, UW Madison, Madison, WI, United States; Peter Ferrazzano, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Pelin Cengiz, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States

Background: Hippocampal interneurons (INs) maintain the excitatory-inhibitory balance essential for learning and memory during brain development. The tyrosine receptor kinase B (TrkB), encoded by Ntrk2, is critical for IN maturation and hippocampal circuit formation. Loss of TrkB in GABAergic INs disrupts network activity and behavior. Our recent studies using snRNA-seq revealed female-specific Ntrk2 upregulation in IN clusters, particularly within the somatostatin (SST) and Lamp5 INs after hypoxia ischemia related brain injury. However, the baseline IN population sizes and how Ntrk2 expression varies across in the IN subtypes during development remains unknown.

Objective: The objective of this study is to determine the population sizes of major hippocampal IN subtypes and their Ntrk2 mRNA expression in order to characterize sex- and subtype-specific expression profiles.

Design/Methods: Naïve C57BL/6 mice at postnatal days (P) 6, 9, 16 and 30 (n=3 per sex and age per time-point) underwent perfusion fixation. Every 4th-7th 35 µm coronal slice was collected throughout the hippocampus for immunohistochemistry (SST, DAPI) and RNAscope (Gad1, Lamp5, Ntrk2) co-labeling (Fig. 1). Design-based stereology was utilized to quantify the hippocampal IN counts and volumes. Ntrk2 mRNA puncta within the INs were semi-quantitatively analyzed from confocal images of the CA1 region obtained from two randomly selected dorsal and ventral hippocampal slices. Cell densities (cell/mm³) and Ntrk2 puncta counts are presented for the SST and Lamp5 INs co-expressing Gad1. Multifactorial ANOVA was used for data analysis.

Results: In the hippocampus at P16, females showed a higher density of Gad1 expressing INs than males, while Lamp5 and SST expressing IN densities were comparable between sexes (Fig. 2). Baseline Ntrk2 mRNA expression within SST expressing INs was higher in males compared to females at P16 (P=0.05). In males, SST expressing INs also showed greater Ntrk2 expression than Lamp5 expressing INs (P=0.02) (Fig.3).

Conclusion(s): These findings reveal sex- and subtype-specific differences in hippocampal INs during early postnatal development. While IN densities are largely similar between sexes at P16, Ntrk2 expression varies by both sex and IN subtype in the CA1 region. Ongoing experiments will extend these analyses to additional developmental stages (P6, P9 and P30), hippocampal subregions (CA3 and dentate gyrus), and will include parvalbumin expressing INs to further define temporal and molecular characteristics of IN development and Ntrk2 expression.

Figure 1. Experimental Design


Figure 2. Gad1 expressing interneuron densities were significantly higher in females compared to males in both the left and right hippocampus at P16. In contrast, SST and Lamp5 expressing interneuron populations showed no sex differences.
A. 5x confocal image of naïve left hippocampus probed for Gad1 (green), Lamp5 (red) mRNA and stained for SST protein (cyan). B. 63x confocal images from the CA1 region of naïve left hippocampus. C. Hippocampal interneuron densities (cell/mm³) in male (M) and female (F) naïve mice at P16. Data are presented as mean ± SEM, n = 3 per sex. Statistics 2- way ANOVA.

Figure 3. The hippocampal SST expressing interneurons have more Ntrk2 mRNA puncta compared to Lamp5 expressing interneurons in male naïve mice at P16.
A. Representative 20x confocal images of the naïve left hippocampus showing Lamp5 (red) and SST (cyan) expressing interneurons probed for Ntrk2 mRNA (magenta). B. Number of Ntrk2 mRNA puncta in hippocampal Lamp5 and SST expressing interneurons of male and female naïve mice at P16. Data are presented as mean ± SEM, n = 3 per sex. Statistics 2- way ANOVA.